Difference between revisions of "Team:SSTi-SZGD/Design"

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Overview: ( background introduction )

The aim of the project this year is to construct recombinant Bacillus subtilis strains that are able to secrete large amount of hyaluronic acid (HA) of both high and low molecular weights. While HA of different molecular mass are already of great commercial values ^[1] , we also proposed to use the low molecular weight HA in the development of a novel form of cosmetic product – HA micro-needles. In designing our project we adhered to three over-arching engineering principles: 1) HA biosynthesis is achieved via a safer, cost effective, and highly productive approach compare with the traditional methods. To achieve this goal, we constructed a recombinant B.subtilits 168 strain that heterogeneously expresses HA synthase for the biosynthesis of HA, given the fact that two HA precursors, GlcUA and GlcNAc, are synthesized indigenously by B. subtilis. In addition, a number of native genes, gtaB, tuaD, glmU, glmS, and glmM, whose gene products work together to regulate the biosynthesis of GlcUA and GlcNAc. Overexpression of these native genes may help improving the accumulation of HA; 2) Low molecular weight HA is biosynthesized by hydrolyzing high-molecular-weight HA in cell cultural environment. A leech hyaluronidase gene (LHayal) encodes LHAase is heterogeneously expressed in the recombinant B.subtilits 168 strain constructed in step 1. LHAase belongs to the hyaluronate 3-glycanohydrolase subgroup and has high substrate specificity and hydrolysis efficiency; 3) To ensure that our HA product has better absorbing efficiency and can be easily applied, we designed a novel HA product—HA micro-needles patch--by using colvantly cross-linked HA hydrogel and HA molecule mixture. Altogether, we divided our project into three modules to achieve these goals collectively.

Module 1 - HA Production

1. HA Introduction and Plasmid construction

2. HA Concentration Elevation

3. Transformation

4. Fed-batch Fermentation and Purification of HA

5. HA Characterization

HA Introduction

Hyaluronic acid (HA) is a mucopolysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerization ^[1] . One of the mainstream industrial HA productions is through streptococcus.zooepidemicus fermentation, which currently facing two challenges, 1) the high risk of pathogenicity and the fewer DNA manipulation techniques available restricted the use of streptococcus.zooepidemicus species, 2) viscoelastic property of HA significantly reduces dissolved oxygen in fermentation, which is the primary obstacle in maintaining normal cell metabolism and improving HA biosynthesis.

Therefore, in order to solve the first issue, our project devoted to construct a food-grade safe (GRAS) strain, B.subtilis , for HA biosynthesis, and to increase production level of HA by regulating some of the precursor genes in the upstream synthetic pathways; for the second issue, we tried to directly produce HA at lower molecular weight with reduced viscosity.

(1) HA bio-synthesis pathway

Studies of HA biosynthesis in prokaryotes have shown that an indigenous pathway for biosynthesis of HA precursors exists in B.subtilis genome, starting from sucrose as a carbon source, two parallel metabolic branches form which eventually, through multiple sugar intermediates, culminate in the synthesis of the two nucleotide sugar substrates, UDP-GlcUA and UDP-GlcNAc ^[2]

In the first set of reactions, α-phosphoglucomutase (pgcA) converts glucose-6-phosphate to glucose-1-phosphate before a phosphate group from UTP is transferred to glucose-1-phosphate by UDP-glucose pyrophosphorylase (gtaB) to produce UDP-glucose. Later, UDP-glucose is oxidised by UDP-glucose dehydrogenase (tuaD) to yield the first HA precursor, UDP-glucuronic acid (UDP-GlcUA). In the second set, glucose-6-phosphate is converted to fructose-6-phosphate by phosphoglucoisomerase (pgi). Once converted, fructose-6-phosphate is tagged with an amino group transferred from a glutamine residue via amidotransferase (glmS) to produce glucosamine-6-phosphate and modified by mutase (glmM) to yield glucosamine-1-phosphate. This intermediate is then sequentially acetylated and phosphorylated by acetyltransferase and pyrophosphorylase(glmU), respectively to yield the second HA precursor, UDP-N-acetylglucosamine (UDP-GlcNAc). Once the two precursors are synthesized, hyaluronic acid synthase (hasA) polymerises the two components in an alternate manner to produce HA polymer ^[2] . It is important to note that HA biosynthesis is an energy-consuming process for the bacteria as several intermediates are also used in cell wall biosynthesis, biomass formation and lactate formation via glycolysis ^[3] . Streptococcus.zooepidemicus has all the require pathways and genes for HA synthesis,however, B.subtilis , however, lacks the crucial hasA gene that is responsible for the final polymerization step in HA synthesis ^[4] .

Fig 1: Hyaluronic acid bio-synthesis pathway in S.zooepidemicus .

(2)Construction of pAX01-HasA plasmid

To construct a HA biosynthetic pathway in B. subtilis 168 , the missing hyaluronic acid synthase encoding gene hasA was amplified from S. zooepidemicus . Heterogenous expression of HasA gene was achieved by an integration vector pAX01 to avoid plasmid loss. The constructed HasA expression cassette, under the control of an inducible promoter PxylA, was integrated at the lacA locus of the B. subtilis 168 genome.

① pAX01 backbone

pAX01 backbone contains two antibiotic resistant genes for different chassis selections (Erm and AmpR), homologous arm lacA which is used for integration of HasA construct into B. subtilis 168 genome, a xylose-induced promoter PxylA originated from B.megaterium , and a repressor xylR regulatory cassette. Promoter PxylA is located within xylose operon, originally to drive the expression of xylA (xylose isomerase coding gene) and xylB (xylulose kinase). xylR with its promoter located at upstream of xylose operon. It encodes xyl repressor which binds to xyl operator in the absence of xylose, repressing transcription activation.

② HasA construct

HasA construct contains a hasA coding sequence in combination with a strong ribosome binding site (RBS). HasA coding sequence is isolated from S.zooepidemicus encodes hyaluronic acid (HA) synthase, which is 419 amino acid long and forms part of the HA synthesis operon in S.zooepidemicus . HasA gene was commercially synthesized and sub-cloned into pAX01 integration vector at the SacII and BamHI restriction sites.

Fig 2：Illustration of HasA construct in pAX01 plasmid

Design of pP43NMK-gtaB-tuaD and pP43NMK-glmU plasmids

TuaD-gtaB construct was commercially synthesized by GBI Genome Service, Shenzhen, and was sub-cloned into shuttle vector pP43NMK at the KpnI and HindIII restriction sites. The expression of operon tuaD-gtaB was under the control of a constitutive promoter P43.

① pP43NMK backbone

p43NMK backbone contains 1) P43 constitutive promoter and an B. subtilis RBS, 2) two antibiotic resistant genes for different chassis selections (Km and AmpR), 3) an E. coli replication origin (ori), 4) M13 fwd and rev universal sequences for sequencing purpose.

② TuaD-gtaB construct

TuaD-gtaB construct contains two gene coding sequences in an operon. TuaD is one of the native genes of B. subtilis, encoding UDP-glucose 6-dehydrogenase and is 461 amino acid in length (also known as: UDP-GlcDH in megaterium); gtaB encodes UTP--glucose-1-phosphate uridylyltransferase in Bacillus megaterium .

Fig 3：Illustration of tuaD-gtaB construct in pP43NMK plasmid

③ glmU construct

glmU construct contains a coding sequence of glmU gene, which is isolated from Mycobacterium encodes UDP-N-acetylglucosamine pyrophosphorylase. GlmU gene was commercially synthesized and sub-cloned into pP43NMK vector at the BamHI and HindIII restriction sites. The constructed glmU expression cassette is under the control of the constitutive promoter P43.

Fig 4：Illustration of glmU construct in pP43NMK plasmid

Transformation

Recombinant vector pAX01-HasA was transformed into the wild-type strain B.subtilis 168 (LB+ 0.5μg/ml erythromycin) to obtain a recombinant B. subtilis strain 168E using chemical transformation method. pP43NMK-tuaD-gtaB and pP43NMK-glmU vectors were then transformed into the recombinant strain B.subtilis 168E (LB+50μg/ml kanamycin), respectively, to study whether overexpression of these precursor genes could elevate HA production. Cultures were grown overnight at 37℃., at shaking. Colony PCR and restriction enzyme digestion of the miniprepped plasmids confirmed that these genes were successfully transformed into B.subtilis .

The primers of colony PCR which is used for verification:
HasA-F: GGTCCATAGGGCTACAAAAG
HasA-R: ACCCTGATGCTTTAGAGGAG
GlmU-F: GGCTGGACAAGGAACGAGAA
GlmU-R: CGTCTCTGCTGTCAAAAGCG
GtaB-TuaD-F: CACAGTAGCGGGTACTGGTTA
GtaB-TuaD-R: TTGGATGCTACCTCAACTTGT

Fed-batch Fermentation of HA , HA Separation and Purification

After transformation was confirmed, single colony was inoculated into 50ml LB medium and grew overnight as a seeded culture. The next day re-inoculated into 50ml MM media (1%v/v) and grew at 37℃ at shaking 160rpm. Xylose (20g/L) was added 2h after inoculation to induce the expression of the hasA gene. Sucrose was exponentially fed at rates of 7.5, 7.5, 15.0, and 10.0g/h/L from 8 h to 12 h. A constant feed rate of 5g h/L was then maintained until the end of fermentation for 48h ^[5] .

The HA sample was released by the addition of 0.1% (w/v) sodium dodecyl sulfonate (SDS), culture supernatant was collected by centrifugation at 10,000g for 10min and filtered through a 0.45um micro-filter membrane ^[6] . HA was precipitated with two volumes of ethanol and incubated at 4°C for 1h. The precipitate was collected by centrifugation at 5000g for 10min. Then re-dissolved in an equal volume of distilled water, the steps were performed three times. Purified HA was then freeze-dried to become powder before subject to characterization.

Fig 5：Demonstration of a freeze-drying machine that freeze-dried HA molecules

HA Characterization

(1) CTAB(cetyltrimethylammonium bromide) method

CTAB is known as a common method for measuring the concentration of the HA. It is a cationic surface active agent which precipitates nucleic acids and acidic polysaccharides under the low ionic strength. At the same time, CTAB forms complexes with proteins and polysaccharides, acidic polysaccharides under the high ionic strength ^[7] .

The nitrogen atoms of CTAB can be paired with the oxygen atoms of carboxyl groups in HA to form insoluble HA-CTAB complexes. Meanwhile, CTAB does not interact with the UDP-gluconic acid (UDP-Gluconate) and UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) which is the monomers of the HA. This method is therefore specific for measuring HA concentration. 2.5g/L CTAB solution was prepared (containing 0.2mol/L NaOH and appropriate amount CTAB powder). 1ml re-dissolved HA powder or standard sample and 2ml CTAB solution was thoroughly mixed in colorimetric dish. The timing was started once the mixing was, and the absorbance was measured at 400nm wavelength at the 10min point. 1ml deionized water with CTAB solution was set as a blank. The standard curve was plot using a series dilution of the standards.

(2) Ubblelohde viscometer method

Ubblelohde viscometer method was used to get a rough idea of the molecular weight of HA, usually HMW-HA. Ubbelohde viscometer is an instrument which uses a capillary based method for measuring viscosity ^[8] . It has a reservoir on one side and a measuring bulb with a capillary on the other. Once liquid is introduced into the reservoir then sucked through the capillary and measuring bulb. Liquid then travels back through the measuring bulb and the time takes for the liquid to pass through two calibrated marks is recorded to calculate the viscosity according to the Hagen-Poiseuille law:

Fig 6: Illustration of an Ubblelohde viscometer and the formula used to viscosity calculation, in which η, ηsp, C, k, β, lnηr are solution viscosity, increased specific viscosity, concentration, huggins constant, kramer constant, and logarithmic viscosity of the capillary respectively. Volume flow measurement through the capillary at a given differential pressure is the fundamental measurement criteria for capillary viscometers. Expressed differently, the viscosity is determined by measuring the time required for a defined liquid volume to flow through a capillary tube determined by the hydrostatic pressure of the liquid. Two marks before and after a ball shaped extension enables measurement the time.freeze-dried HA molecules

The standard HA solution was prepared by dissolving 0.1g of HA in 100ml 0.2M NaCl solution, then diluted into different concentrations: 0.02g/100ml, 0.03g/100ml, 0.04g/100ml, and 0.05g/ml. After cleaning of the viscometer, solutions were injected to flow through the tubing and the device was inserted into a constant temperature bath. The solution was measured according to Ubblelohde viscometer instruction manual. This measurement was performed in duplicates and repeated three times under 25℃.

Module 2 - LHAase Construction

1. Hyaluronidase Introduction

2. Plasmid Construction

3. Transformation and Expression

4. LHAase Characterization

5. LMW-HA Synthesis

(1) LHAase introduction and Hydrolytic pathway of HA using LHAase

Hyaluronidase（HAase）is denoted to a large class of enzymes that predominantly degrade HA ^[9] . HAases widely exist in eukaryotes and prokaryotes, and are important physiological active substances participating in many physiological activities ^[10] . Based on substrate specificity and hydrolysis products, HAases are commonly grouped into three families: the first is hyaluronate lyases (EC 4.2.2.1, streptococcus.zooepidemicus hyaluronate lyase), which is a common source of commercial HAase production but may contain endotoxins. It hydrolyzes HA on the β-1, 4 glycoside bond to generate 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-D -glucose.The second group is hyaluronate 4-glycanohydrolases (EC 3.2.1.35, Bovine testicular hyaluronidase, BTH). Commercial BTH has been widely used in clinical medicine, and its hydrolysis mechanism has been studied extensively. Besides being expensive in material source (bovine testes), BTH hydrolyzes HA by cutting the β-1, 4 glycoside bonds to produce mainly four sugar. Also it could hydrolyze chondroitin and has transglycosidation. The Third group is hyaluronate 3-glycanohydrolases (EC 3.2.1.36, Leech HAase).

Compared with BTH and streptococcus.zooepidemicus HAases, leech HAase has high substrate specificity and can produce a narrow-spectrum of products. It degrades HA by crackingβ- 1, 3 - glycosidic bond, results in the reduction of side with glucuronic acid sugar fragments. The end products are mainly four and six sugars. In addition, the use of recombinant leech HAase does not pose any risk of animal cross-infection and it has no transglycosidation action ^[11] . Therefore, high-level production of recombinant leech HAase would be of great significance for both clinical medical treatments (such as surgery, ophthalmology and internal medicine), and producing narrow-spectrum HA oligosaccharides at the industrial scale.

Fig 7: Illustration of Hydrolytic pathway of HA by LHAase

(2) LHAase introduction

We cloned the first leech HAase-encoding gene, LHyal, into the recombinant HA-producing B.subtilis strain 168E in order to achieve a cell factory synthesizing low molecular weight HA. LHyal gene (Genebank NoKJ026763) encodes LHAase (Mw=58kD), belongs to the hyaluronate 3-glycanohydrolases family. HA was hydrolyzed to oligosaccharide by hydrolyzingβ-1.3 glucosidic bonds by LHAase in a non-processive endolytic mode ^[12] . LHyal has superior substrate specificity and no transglycosidase activity compare with other two group of hyaluronidase. In particular, leech HAase is unable to degrade chondroitin or chondroitin sulfate. Although mammalian HAase has been widely used as a drug diffusion agent, such HAase activity is susceptible to heparin inhibition. In comparison, leech HAase activity is not affected by heparin, therefore it possesses more medical value in clinic and other medical aspects ^[13] .

LHAase Plasmid Construction

In order to identify the best gene expression strategy for LHAase, two expression vectors were selected: integration vector pDG1730 and shuttle vector pMA0911. LHyal expression construct was commercially synthesized by BGI Genome Services and sub-cloned into vectors pDG1730 or pMA0911 respectively.

① Design of LHAase expression vector

p43NMK backbone contains 1) P43 constitutive promoter and an B. subtilis RBS, 2) two antibiotic resistant genes for different chassis selections (Km and AmpR), 3) an E. coli replication origin (ori), 4) M13 fwd and rev universal sequences for sequencing purpose.

The LHyal expression construct contains 1) a B.subtilis constitutive promoter PlepA, 2) 6xHis-tag, 3), a strong ribosome binding site (RBS) which is a shine-Dalgarno sequence from gsiB gene, 4) a signal peptide of AmyX that is derived from a-Amylase gene from B.amyloliquefaciens , and 5) LHyal coding sequence Leech.

Fig 8: Illustration of LHyal construct in pDG1730 plasmid

Promoter：

PlepA is a strong constitutive promoter which is derived from the bicistronic operon. One of the expressed proteins in the operon is protein LepA. This protein plays an important role during the translation as it can move the mRNA-tRNA complex one step back in the ribosome which is expected to improve the fidelity of translation.

6 His-tag：

Currently His-tags have been extensively applied for recombinant protein expression. Based on our literature search, 6His-tag seems to provide enough spacing for protein folding to prevent crystallization or misfolding of peptide chain, and it is a commonly used in protein purification by chromatography.

RBS：

The strong ribosome binding site (RBS) is a shine-Dalgarno sequence from gsiB gene. It can lead to a pronounced stimulation of expression when placed upstream of a variety of genes, and significant increase in the translation of the genes is observed.

Signal peptide:

Bacillus subtilis is a well-known chassis with highly active protein secretion system, There are mainly two protein export pathways availibale in B.subtilis: Sec-dependent translocation pathway and twin-arginine translocation (Tat) pathway ^[14] . Compare with Sec pathway, tat pathway has an intrinsic advantage of being able to transport folded proteins across the cytoplasmic membrane and without the requirement ATP hydrolysis, this helps retaining protein function extracellularly ^[15] . Tat pathway has a signature twin-arginine (RR) motif located at the border of the N-terminal domain and the hydrophobic region. AmyX signal peptide belongings to tat pathway. In our project AmyX was fused in N-terminal of the LHyal gene.

pDG1730 plasmid backbone：

contains Bsu-amyE homologous arm sequences that help LHyal construct to be integrated into the amyE locus of B.subtilis genome by double-crossover integration. There is also an ori sequence for replication in E. Coli , three antibiotic resistant genes for E.coli and B. subtilis selections (Amp, Erm and Spc), and a T1 terminator sequence. the whole construct was sub-cloned into the backbone at the BamHI and HindIII restriction sites.

pMA0911 plasmid backbone：

consists of two antibiotic resistant genes which are used for different chassis selections (Kane, Amp) and an E. coli ori sequence. The construct was sub-cloned into the backbone at the NdeI and BamHI restriction sites.

Transformation and Expression

① Transformation

pDG1730-LHyal construct vector was transformed into B.subtilits 168 by chemical transformation method and grew on LB agar+antibiotic at 37℃overnight. Positive selection of integration was performed with spectinomycin at 100μg/ml and negative selection of single crossover integration events with erythromycin at 0.5μg/ml. Colony PCR for verifying part integration were realized using Taq polymerase PCR system (Takara).

The primers for colony PCR polymerization:
LHAase-F: ATGAAAGAGATCGCGGTGA
LHAase-R: TTATTTTTTGCAGGCTTC

② Fed-batch fermentation

Once colonies contain recombinant bacteria were identified, single colony was streaked out into a starter 1ml LB culture (containing 50ug/ml erythromycin) and grew at 30℃, 220rpm, overnight culture was inoculated into 50ml LB medium+ 50ug/ml erythromycin (1% v/v) and grew for 12 h at shaking before fresh 50ml LB medium+ 50ug/ml erythromycin was added. Every 12 h, fresh 50ml LB with antibiotic was fed until 48 h later a 200ml culture was achieved. Cell density was measured every 12 h. After the final feed, when OD600 reached ~2.5, the bacterial cells were removed by centrifugation and filtered through a 0.45um micro-membrane. Culture supernatant was freeze-dried to become powder before further purification.

③ LHAase purification

LHAase was isolated and purified from freeze-dried supernatant powder by Ni-NTA spin columns (Biorad Cat.no. 31314). Briefly, dried powder was dissolved into deionized water and added 5ul RNase then the 630ul enzyme liquid was added to Ni-NTA column [QIAGEN Co Ltd.Cat NO31314] for 5min, the non-target proteins were eluted by using 600ul NPI-20 (50mM NaH2PO4, 300mMNaCl, 20mM imidazole) and was centrifugated 2 times, supernatant was collected by 890g centrifugation. Target protein was then eluted by using 300ul NPI-500 (50mM NaH2PO4, 300mMNaCl, 500mM imidazole) and supernatant collected by centrifugation. Purified enzyme was dialyzed (Regenerated cellulose dialysis membrane MWCO 8000-14000) against 50mM phosphate buffer (pH:6-7.4) overnight before HA was freeze-dried. Purified HA powder was analyzed by SDS-Polyacrylamide gel electrophoresis (PAGE) and enzymatic analyses.

④ SDS-PAGE

Appropriate freeze-dried LHAase powder was mixed with 6xloading buffer (30mM EDTA,36%(v/v) glycerol, 0.05%(w/v), xylene cyanol FF, and 0.05% (w/v) Bromophenol blue). Sample was denatured by boiling for 10 min, and then loaded into the gel. Electrophoresis was performed at 150V, 80mA for 1 h. Gel was stained and destained before photography.

Enzymatic Characterization of LHAase

① Agarose Plate Assay by using HA as a substrate

LHAase activity was confirmed using the simple plate assay ^[16] . The assay plate was prepared with 1mg/ml HA, 0.75g agarose, 50mM sodium citrate (pH5.5) buffer. 150ul sample, control or standard were injected into the cylindrical holes (3mm in diameter) on the agarose plates, incubated at 37℃ for 10h and covered with 10%(w/v) cetylpyridinium chloride. The formation of a distinct clear halo around the hole indicates HAase activity. Standard enzyme solution (hyaluronidase extracted from bovine testis) was set as positive control, heat-inactivated enzyme solution was set as negative control.

② Enzyme Linked Immunosorbent Assay (ELISA)

ELISA is a very sensitive immunochemical technique used to assess the presence of specific protein (antigen or antibody) in the given sample and its quantity. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. LHAase activity was studied using a microorganism HA ELISA kit (Tongwei Ltd, Shanghai, Cat.no tw045410). Firstly, 40μl standard and 10ul sample were mixed and loaded into a 96 well microplate. No sample is added to the blank well. 100μl enzyme-conjugate was added to standard wells and sample wells except the blank well, then cover with an adhesive strip and incubate for 60min at 37℃. The microplate was washed 4 times using 20x eluent. Substrate A (50μl) and Substrate B (50μl) were added to each well. Gently mix and incubate for 15m at 37℃, stop solution was added to each well to induce color changes. The Optical Density (O.D.) is read at 450 nm using a microtiter plate reader within 15min. This assay was performed in duplicates and repeated three times.

③ DNS reducing sugar method

LHAase activity analysis was further examined by measuring the amount of reducing sugar liberated from HA with the 3,5-dinitrosalicylic acid (DNS) colorimetric spectrophotometric ^[17] . One unit of enzymatic activity was tentatively defined as equal to the reducing power of glucuronic acid (glucose equivalents in micrograms) liberated per hour from HA at 38℃ and pH5.5. Specific activity was defined as units of enzyme per mg of protein.

In our experiment, the enzymatic reaction contained 1.6 mg/ml of HA as a substrate and an appropriate amount of LHAase in 50 mM pH 5.5 citrate buffer in a total volume of 1 ml, and was incubated at 38℃ for 10 min. The reaction was stopped by immersion in boiling water for 5 min and before adding DNS solution and further boiled for 15min to induce color changes. Standard enzyme solution (hyaluronidase extracted from bovine testis) was set as positive control, heat-inactivated enzyme solution was set as negative control.

(1)LMW-HA Synthesis

Currently, low molecular weight HA has attracted considerable attention because of its potential applications attributed to its unique biological properties, including the stimulation of fibroblast proliferation, collagen synthesis, and potential effects in eliminating certain cancer cells ^[18] . Compare with HMW-HAs, LMW-HAs can be readily absorbed by human body and contribute to the biosynthesis of HMW-HA ^[19] . With its important values in medical and cosmetics, it is beneficial to directly obtain low-molecular weight HA from microbial fermentation via LHAase hydrolysis.

① Transformation

pDG1730-LHAyal and pMA0911-LHAyal vectors were transformed into the recombinant strain B.subtilis 168E , respectively, and selected by antibiotic kanamycin to study the direct biosynthesis of LMW-HA in B.subtilis 168E-LHAase . Colony PCR and restriction enzyme digestion confirmed that these vectors was successfully transformed into B.subtilis 168E-LHAase .

The primers of colony PCR which is used for verification:

HasA-F: GGTCCATAGGGCTACAAAAG
HasA-R: ACCCTGATGCTTTAGAGGAG
LHAase-F: ATGAAAGAGATCGCGGTGA
LHAase-F: ATGAAAGAGATCGCGGTGA

② Fermentation and HA purification

Refer to HA Fed-batch fermentation of HA and HA separation and purification

(2)HA characterization

① CTAB

Refer to CTAB (cetyltrimethylammonium bromide) method

② HA molecular weight easurement--GPC-RI-MALS

Gel chromatography (GPC-RI-MALS) is an effective determination of molecular weight of polysaccharides, especially for low-molecular-weight HA that cannot be directly measured using a viscometer ^[20] . The principle is: when samples of different molecular weights pass through gel column, the distance and the time of the process will be changed according to different molecular weight of HA, therefore different substances can be separated. Then the molecular weight and abundance of elements can be detected by differential detector and multiple angle laser light scattering.

GPC-RI-MALS service was provided by Sanshu Biotechnology Co, Shanghai. Purified HA samples were sent to them. To test our HA samples, the mobile phase of the measurement is NaNO3, 10mg of HA sample was dissolved respectively by adding 1.5ml NaNO3, then the mixtures were centrifuged (12000rpm/10min) and filtered through a 0.22um filter membrane. Ultimately 100ul sample was analyzed by gel chromatography.

Module 3- HA Micro-needle Design

1. Micro-needle Concept

2. Fabrication of cHA Hydrogel Particles

3. Preparation of the Micro-needle Mold

4. Fabrication of HA-cHA-Micro-needles

5. Water Solubility Test

Micro-needle Design

At present, the preparation of micro-needles with natural and degradable polymer bio-materials has become a hot topic. In this project we designed a new type of micro-needles by using HA products from previous section.

Cross-link is widely used in dermal filler preparation by linking one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers ^[21] . In polymer chemistry, "cross-linking" usually refers to the use of cross-linking agents to promote a change in the polymers' physical properties.。

At present, the mainstream chemical cross-linking agents for cosmetic HA implant preparations are diethylsulfone (DVS) and 1, 4-butanediol diglycidyl ether (BDDE) ^[22] . DVS, although widely used in R&D, has high cytotoxicity. Not only this toxic substance is likely to be accumulated in the body after implantation, but also it may affect normal tissue growth due to calcification of the implant. In comparison, BDDE is the most commonly used cross-linking agent, it is biodegradable, much less toxic and more reactive than DVS, thereby safer for biomedical applications. Cross-linked with HA using BDDE making it more stable, less susceptible to enzymatic degradation, and with increased mechanical strength ^[23] . Previous studies ^[24] showed that under alkaline condition, the opening of BDDE rings during reaction reacts with the OH group of HA to produce crosslinked network (hydrogel).

Micro-needles made with cross-linked HA has better swelling ability and slow bio-degradation that could result to a prolonged effectiveness of the dermal filler, which has the potential to replace surgical approach or injection of HA for anti-wrinkle treatment. In addition, we performed water solubility test in water to understand whether cross-linking, solidifying and curing process could affect HA solubility.

Fig9: Hyaluronic acid cross-linking

Fabrication of cHA Hydro-gel Particles

Cross-linked HA (cHA) reagent solution was prepared by mixing 200μL of 1,4-butanediol diglycidyl ether (BDDE) into 9.8mL of 0.25mol/L NaOH (pH13). Approximately 1.0g of HA powder was added to the cHA reagent solution and stirred well, and thoroughly mixed at 40°C for 5 hours. The prepared hydrogel was grounded, squeezed out and screened with a 170 mesh sieve to get particles having a diameter of less than 90μm ^[25] .

Preparation of The Micro-needle Mold

The micro-molds made of resin were prepared by 3D-printed technology provided by the Engineering Department of our school. The needle cavities are cone shape, with 6500um in depth and 2500um in diameter, and on the micro-molds are patterned into 3 × 3 (round shape) and 10 × 10 (rectangular shape).

Fig10：Illustration of micro-needle molds

Fabrication of HA-cHA-micro-needles

The HA-cHA-micro-needles was prepared by mixing different proportions of uncross-linked HA powder and cross-linked HA (cHA) powder to become mixtures, the ratios tested were 1:0, 1:1, and 5:1 (HA-cHA) ^[26] . The mixtures were dissolved in ultrapure water and a 20% (w/v) viscous polymer solution was stirred at room temperature under a magnetic stirrer for the production of homogenous suspension. The homogenous suspension was poured into the mold, and centrifuged at 10,000 rpm for 10 min to ensure the full loading to the needle cavities. Excess liquid was removed by using a tape. This step was repeated twice until the needle cavity was full, and then the viscous polymer solution was poured onto the surface of the mold, any residual solution remaining at the edge of the mold was removed by a blade. After curing overnight at room temperature, micro-needle was successfully prepared by separating from the mold using a blade.

Fig11: Flow chart of preparation of microneedle

Water Solubility Measurement

Cured micro-needles into water at room temperature for 10 min to study water solubility property. Dissolving status was recorded.

References

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[2] Sloma, A., R. Behr. 2003. Methods for producing hyaluronan in a recombinant host cell. World patent application WO03/054163.

[3] Crater, D. L., and I. van de Rijn. 1995. Hyaluronic acid synthesis operon (has) expression in group A streptococci. [J]. Biol. Chem. 270:18452-18458.

[4] Widner, B., M. Thomas.2000. Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes. [J]. Microbiol. Biotechnol.25:204-212

[5] Meng, G.,Fütterer, K.,2003.Structural frame work of fructosyl transfer in Bacillus subtilis levan sucrase.Nat.Struct.Mol.Biol.10,935–941.

[6] Peng Jin, Guocheng Du. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers [J].Scientific Reports, 2014: 1-2

[7] Yong-Hao C, Qiang W, Establishment of CTAB Turbidimetric method to determine hyaluronic acid content in fermentation broth [J] Carbohydrate Polymers 78 (2009) 178–181

[8] Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers[J].Scientific Reports, 2014: 1-2

[9] Ferguson, E. L., Roberts, J.Griffiths, P. C., & Thomas, D. W. (2011). Eval-uation of the physical and biological properties of hyaluronan and hyaluronan fragments. International Journal of Pharmaceutics, 420, 84–92.

[10] Kogan, G., Soltes, L., Stern, R., & Gemeiner, P. (2007). Hyaluronic acid: A natural biopolymer with a broad range of biomedical and industrial applications.Biotechnology Letters, 29, 17–25.

[11] Jin, P., Kang, Z., Zhang . (2014). High-yield novel leechhyaluronidase to expedite the preparation of specific hyaluronan oligomers.Scientific Reports, 4, 1–8.

[12] Linker, A., Hoffman, P. (1957). The hyaluronidase of the leech: Anendoglucuronidase. Nature, 180, 810–811.Linker, A., Meyer, K., & Hoffman, P. (1960).

[13] Nermeen S.El-Safory The production of hyaluronate oligosac-charides by leech hyaluronidase and alkali. Journal of Biological Chemistry, 235,924–927.

[14] Sargent.F (2007) The twin-arginine transport system:moving folded proteins across membranes. Biochem SocTrans 35, 835–847.

[15] Jongbloed JD, Grieger U,(2004) Twominimal Tat translocases in Bacillus. Mol Microbiol 54,1319–1325.

[16] Richman, P. G. & Baer, H. A convenient plate assay for the quantitation of hyaluronidase in Hymenoptera venoms. Anal. Biochem. 109, 376–381 (1980).

[17] Jin,P.,Kang,Z.,Zhang .2014.High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers.[J].4,4471.

[18] Ghatak,S.,Misra,S.,Toole,B.P.,2002.Hyaluronan oligosaccharides inhibit anchorage independent growth of tumor cells by suppressing the phosphoinositide 3-kinase[J].277,38013–38020.

[19] Boltje,T.J.,Buskas,T.,Boons,G.J.,2009.Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research.Nat.Chem.1,611–622.

[20] Tranchepain, F. Deschrevel, B.et al. (2006). A complete set of hyaluronan fragments obtainedfrom hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluro-nan mass distribution by simple HPLC devices. Analytical Biochemistry, [J]348,232–242.

[21] Ghosh K, Shu XZ, Mou R, Lombardi J, Prestwich GD, Rafailovich MH, et al., Rheological characterization of in situ cross-linkable hyaluronan hydrogels, Biomacromolecules, 2005;6(5):2857-65.

[22] K. De Boulle, R. Glogau, A Review of the Metabolism of 1,4-Butanediol Diglycidyl Ether-Crosslinked Hyaluronic Acid Dermal Fillers[J] 39 (2013) 1758.

[23] M. Al-Sibani, A. Al-Harrasi.Study of the effect of mixing approach on cross-linking efficiency of hyaluronic acid-based hydrogel cross-linked with 1,4-butanediol diglycidyl ether.[J] 91 (2016) 131.

[24] B. Yang, X. Guo, H.Zang,Determination of modification degree in BDDE-modified hyaluronic acid hydrogel by SEC/MS. [J]131 (2015) 233.

[25] Jeong YI, Kim ST, Jin SG,et al., Cisplatin incorporated hyaluronic acid nanoparticles based on ion complex formation, Journal of pharmaceutical sciences, 2008;97(3):1268-76.

[26] Vauthier C, Bouchemal K. Methods for the preparation and manufacture of polymeric
nanoparticles, Pharmaceutical research, 2009;26(5):1025-58.

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@@ Line 272: / Line 272: @@
 				<!--Module 2 - LHAase Construction-->
 				<section class="col-xs-12 col-sm-10">
-					<p class="title">Module 2 - LHAase Construction</p>
+					<p class="title">Module 2 - LHAase Construction<!--{cn}第2模块 - LHAase构造--></p>
 					<div class="content">
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span class="active">Hyaluronidase Background</span>
+							<span class="active">1. Hyaluronidase Introduction<!--{cn}1.透明质酸水解酶介绍--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>LHAase Plasmid Construction</span>
+							<span>2. Plasmid Construction<!--{cn}2.质粒构建--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>LHAase Transformation and Expression</span>
+							<span>3. Transformation and Expression<!--{cn}3.转化和表达--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>Enzymatic Characterization of LHAase</span>
+							<span>4. LHAase Characterization<!--{cn}4.LHAase的酶学表征--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>LMW-HA Synthesis</span>
+							<span>5. LMW-HA Synthesis<!--{cn}5.LMW-HA合成--></span>
 						</div>
 						<div class="clearfix"></div>
@@ Line 294: / Line 294: @@
 							<!--One-->
 							<div class="One">
-								<p class="subtitle">① HAase introduction and Hydrolytic pathway of HA using LHAase</p>
+								<p class="subtitle">(1) LHAase introduction and Hydrolytic pathway of HA using LHAase<!--{cn}使用LHAase对HA进行LHAase引入和水解途径--></p>
 								<p>
-									Hyaluronidase（HAase）is denoted to a large class of enzymes that predominantly degrade HA
+									Hyaluronidase（HAase）is denoted to a large class of enzymes that predominantly degrade HA<!--{cn}透明质酸酶（HAase）表示主要降解HA的一大类酶-->
 									<sup>[9]</sup>
-									. HAases widely exist in eukaryotes and prokaryotes, and are important physiological active substances participating in many physiological activities
+									. HAases widely exist in eukaryotes and prokaryotes, and are important physiological active substances participating in many physiological activities<!--{cn}。 HAase广泛存在于真核生物和原核生物中，是参与许多生理活动的重要生理活性物质-->
 									<sup>[10]</sup>
-									. Based on substrate specificity and hydrolysis products, HAases are commonly grouped into three families: the first is hyaluronate lyases (EC 4.2.2.1, streptococcus.zooepidemicus  hyaluronate lyase), which is a common source of commercial HAase production but may contain endotoxins. It hydrolyzes HA on the β-1, 4 glycoside bond to generate 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-D -glucose.The second group is hyaluronate 4-glycanohydrolases (EC 3.2.1.35, Bovine testicular hyaluronidase, BTH). Commercial BTH has been widely used in clinical medicine, and its hydrolysis mechanism has been studied extensively. Besides being expensive in material source (bovine testes), BTH hydrolyzes HA by cutting the β-1, 4 glycoside bonds to produce mainly four sugar. Also it could hydrolyze chondroitin and has transglycosidation. The Third group is hyaluronate 3-glycanohydrolases (EC 3.2.1.36, Leech HAase).
+									. Based on substrate specificity and hydrolysis products, HAases are commonly grouped into three families: the first is hyaluronate lyases (EC 4.2.2.1, <!--{cn}。 基于底物特异性和水解产物，HAase通常分为三个家族：第一个是透明质酸裂解酶（EC 4.2.2.1，-->
+									<i>streptococcus.zooepidemicus<!--{cn}兽疫链球菌--></i>
+									  hyaluronate lyase), which is a common source of commercial HAase production but may contain endotoxins. It hydrolyzes HA on the β-1, 4 glycoside bond to generate 2-acetamido-2-deoxy-3-O-(β-D-gluco-4-enepyranosyluronic acid)-D -glucose.The second group is hyaluronate 4-glycanohydrolases (EC 3.2.1.35, Bovine testicular hyaluronidase, BTH). Commercial BTH has been widely used in clinical medicine, and its hydrolysis mechanism has been studied extensively. Besides being expensive in material source (bovine testes), BTH hydrolyzes HA by cutting the β-1, 4 glycoside bonds to produce mainly four sugar. Also it could hydrolyze chondroitin and has transglycosidation. The Third group is hyaluronate 3-glycanohydrolases (EC 3.2.1.36, Leech HAase).<!--{cn}，透明质酸裂解酶），它是商业HAase生产的常见来源但可能含有内毒素。 它在β-1,4糖苷键上水解HA以产生2-乙酰氨基-2-脱氧-3-O-（β-D-葡糖-4-酮吡喃糖醛酸）-D-葡萄糖。第二组是透明质酸4- 甘氨酸水解酶（EC 3.2.1.35，牛睾丸透明质酸酶，BTH）。 商用BTH已广泛应用于临床医学，其水解机理已得到广泛研究。 除了材料来源（牛睾丸）昂贵外，BTH通过切割β-1,4糖苷键水解HA，主要产生四糖。 它还可以水解软骨素并具有转糖苷作用。 第三组是透明质酸3-甘氨酸水解酶（EC 3.2.1.36，Leech HAase）。-->
 								</p>
 								<p>
-									Compared with BTH and streptococcus.zooepidemicus  HAases, leech HAase has high substrate specificity and can produce a narrow-spectrum of products. It degrades HA by crackingβ- 1, 3 - glycosidic bond, results in the reduction of side with glucuronic acid sugar fragments. The end products are mainly four and six sugars. In addition, the use of recombinant leech HAase does not pose any risk of animal cross-infection and it has no transglycosidation action
+									Compared with BTH and <!--{cn}与BTH和-->
+									<i>streptococcus.zooepidemicus<!--{cn}兽疫链球菌--></i>
+									  HAases, leech HAase has high substrate specificity and can produce a narrow-spectrum of products. It degrades HA by crackingβ- 1, 3 - glycosidic bond, results in the reduction of side with glucuronic acid sugar fragments. The end products are mainly four and six sugars. In addition, the use of recombinant leech HAase does not pose any risk of animal cross-infection and it has no transglycosidation action<!--{cn}HAases相比，水蛭HAase具有高底物特异性并且可以产生窄谱的产物。 它通过裂解β-1,3-糖苷键降解HA，导致葡萄糖醛酸糖片段的侧面减少。 最终产品主要是四糖和六糖。 此外，重组水蛭HAase的使用不会造成动物交叉感染的任何风险，并且没有转糖苷作用-->
 									<sup>[11]</sup>
-									. Therefore, high-level production of recombinant leech HAase would be of great significance for both clinical medical treatments (such as surgery, ophthalmology and internal medicine), and producing narrow-spectrum HA oligosaccharides at the industrial scale.
+									. Therefore, high-level production of recombinant leech HAase would be of great significance for both clinical medical treatments (such as surgery, ophthalmology and internal medicine), and producing narrow-spectrum HA oligosaccharides at the industrial scale.<!--{cn}。 因此，重组水蛭HAase的高水平生产对于临床医学治疗（例如手术，眼科和内科）以及以工业规模生产窄谱HA寡糖将具有重要意义。-->
 								</p>
 								<div class="Cimg col-xs-12 col-sm-6">
-									<img src="https://static.igem.org/mediawiki/2018/thumb/0/02/T--SSTi-SZGD--Design_twoinfo_one.png/750px-T--SSTi-SZGD--Design_twoinfo_one.png"/>
+									<img src="https://static.igem.org/mediawiki/2018/0/02/T--SSTi-SZGD--Design_twoinfo_one.png"/>
 									<p>
-										Fig 7: illustration of Hydrolytic pathway of HA by LHAase
+										Fig 7: Illustration of Hydrolytic pathway of HA by LHAase<!--{cn}图7：LHAase水解酶对HA的水解途径的说明-->
 									</p>
 								</div>
-								<p class="subtitle">② LHAase introduction</p>
+								<p class="subtitle">(2) LHAase introduction<!--{cn}（2）LHAase介绍--></p>
 								<p>
-									We cloned the first leech HAase-encoding gene, LHyal, into the recombinant HA-producing B.subtilis strain 168E in order to achieve a cell factory synthesizing low molecular weight HA. LHyal gene (Genebank NoKJ026763) encodes LHAase (Mw=58kD), belongs to the hyaluronate 3-glycanohydrolases family. HA was hydrolyzed to oligosaccharide by hydrolyzingβ-1.3 glucosidic bonds by LHAase in a non-processive endolytic mode
+									We cloned the first leech HAase-encoding gene, LHyal, into the recombinant HA-producing <!--{cn}我们将第一个水蛭HAase编码基因LHyal克隆到重组HA产生-->
+									<i>B.subtilis strain 168E<!--{cn}枯草芽孢杆菌菌株168E--></i>
+									 in order to achieve a cell factory synthesizing low molecular weight HA. LHyal gene (Genebank NoKJ026763) encodes LHAase (Mw=58kD), belongs to the hyaluronate 3-glycanohydrolases family. HA was hydrolyzed to oligosaccharide by hydrolyzingβ-1.3 glucosidic bonds by LHAase in a non-processive endolytic mode<!--{cn}中，以实现合成低分子量HA的细胞工厂。 LHyal基因（Genebank NoKJ026763）编码LHAase（Mw = 58kD），属于透明质酸3-甘氨酸水解酶家族。 通过LHAase以非进行性内溶解模式水解β-1.3糖苷键将HA水解为寡糖-->
 									<sup>[12]</sup>
-									. LHyal has superior substrate specificity and no transglycosidase activity compare with other two group of hyaluronidase. In particular, leech HAase is unable to degrade chondroitin or chondroitin sulfate. Although mammalian HAase has been widely used as a drug diffusion agent, such HAase activity is susceptible to heparin inhibition. In comparison, leech HAase activity is not affected by heparin, therefore it possesses more medical value in clinic and other medical aspects
+									. LHyal has superior substrate specificity and no transglycosidase activity compare with other two group of hyaluronidase. In particular, leech HAase is unable to degrade chondroitin or chondroitin sulfate. Although mammalian HAase has been widely used as a drug diffusion agent, such HAase activity is susceptible to heparin inhibition. In comparison, leech HAase activity is not affected by heparin, therefore it possesses more medical value in clinic and other medical aspects<!--{cn}。 与其他两组透明质酸酶相比，LHyal具有优异的底物特异性和无转糖苷酶活性。 特别是，水蛭HAase不能降解软骨素或硫酸软骨素。 尽管哺乳动物HAase已被广泛用作药物扩散剂，但这种HAase活性易受肝素抑制。 相比之下，水蛭HAase活性不受肝素的影响，因此在临床和其他医学方面具有更高的医学价值-->
 									<sup>[13]</sup>
-									.
+									.<!--{cn}。-->
 								</p>
 							</div>
@@ Line 325: / Line 331: @@
 							<!--Two-->
 							<div class="Two">
-								<p class="subtitle">① Design of pP43NMK-gtaB-tuaD and pP43NMK-glmU plasmids</p>
+								<p class="subtitle">LHAase Plasmid Construction<!--{cn}LHAase质粒介绍--></p>
 								<p>
-									In order to identify the best gene expression strategy for LHAase, two expression vectors were selected: integration vector pDG1730 and shuttle vector pMA0911. LHyal expression construct was commercially synthesized by BGI Genome Services and sub-cloned into vectors pDG1730 or pMA0911 respectively.
+									In order to identify the best gene expression strategy for LHAase, two expression vectors were selected: integration vector pDG1730 and shuttle vector pMA0911. LHyal expression construct was commercially synthesized by BGI Genome Services and sub-cloned into vectors pDG1730 or pMA0911 respectively.<!--{cn}为了鉴定LHAase的最佳基因表达策略，选择两种表达载体：整合载体pDG1730和穿梭载体pMA0911。 LHyal表达构建体由华大基因商业合成，并分别亚克隆到载体pDG1730或pMA0911中。-->
 								</p>
-								<p class="subtitle">② pP43NMK backbone</p>
+								<p class="subtitle3">① Design of LHAase expression vector<!--{cn}①LHAase表达载体的设计--></p>
 								<p>
-									p43NMK backbone contains 1) P43 constitutive promoter and an B. subtilis RBS, 2) two antibiotic resistant genes for different chassis selections (Km and AmpR), 3) an E. coli. replication origin (ori), 4) M13 fwd and rev universal sequences for sequencing purpose.
+									p43NMK backbone contains 1) P43 constitutive promoter and an <!--{cn}p43NMK骨架含有1）P43组成型启动子和-->
+									<i>B. subtilis<!--{cn}枯草芽孢杆菌--></i>
+									 RBS, 2) two antibiotic resistant genes for different chassis selections (Km and AmpR), 3) an <!--{cn}RBS，2）两种抗生素抗性基因，用于不同的底盘选择（Km和AmpR），3）-->
+									<i>E. coli<!--{cn}大肠杆菌--></i>
+									 replication origin (ori), 4) M13 fwd and rev universal sequences for sequencing purpose. <!--{cn}的复制起点（ori），4）M13 fwd和rev通用序列用于测序目的。-->
 								</p>
 								<p>
-									<span class="subtitle3">The LHyal expression </span>
+									The LHyal expression<!--{cn}LHyal的表达-->
-									construct contains 1) a B.subtilis constitutive promoter PlepA, 2) 6xHistag, 3), a strong ribosome binding site (RBS) which is a shine-Dalgarno sequence from gsiB gene, 4) a signal peptide of AmyX that is derived from a-Amylase gene from B.amyloliquefaciens, and 5) LHyal coding sequence Leech.
+									construct contains 1) a <!--{cn}构建体包含1）-->
+									<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
+									 constitutive promoter PlepA, 2) 6xHis-tag, 3), a strong ribosome binding site (RBS) which is a shine-Dalgarno sequence from gsiB gene, 4) a signal peptide of AmyX that is derived from a-Amylase gene from
+									<!--{cn}组成型启动子PlepA，2）6His标签，3），强核糖体结合位点（RBS），它是来自gsiB基因的shine-Dalgarno序列，4）AmyX的信号肽，来源于 - 来自-->
+								<i>B.amyloliquefaciens<!--{cn}谷氨酸非依赖型的γ-PGA合成菌--></i>
+								 , and 5) LHyal coding sequence Leech.<!--{cn}的淀粉酶基因，和5）LHyal编码序列Leech。-->
 								</p>
 								<div class="img col-xs-12">
-									<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SSTi-SZGD--Design_twoinfo_two.png"/>
+									<img src="https://static.igem.org/mediawiki/2018/5/54/T--SSTi-SZGD--Design_twoinfo_two1.png"/>
 									<p>
-										Fig 8: illustration of LHyal construct in pDG1730 plasmid
+										Fig 8: Illustration of LHyal construct in pDG1730 plasmid<!--{cn}图8：pDG1730质粒中LHyal构建体的图示-->
 									</p>
 								</div>
 								<p>
-									<span class="subtitle3">Promoter: </span>
+									<p class="subtitle">Promoter： <!--{cn}启动子：--></span></p>
-									PlepA is a strong constitutive promoter which is derived from the bicistronic operon. One of the expressed proteins in the operon is protein LepA. This protein plays an important role during the translation as it can move the mRNA-tRNA complex one step back in the ribosome which is expected to improve the fidelity of translation.
+									PlepA is a strong constitutive promoter which is derived from the bicistronic operon. One of the expressed proteins in the operon is protein LepA. This protein plays an important role during the translation as it can move the mRNA-tRNA complex one step back in the ribosome which is expected to improve the fidelity of translation.<!--{cn}PlepA是一种强组成型启动子，来源于双顺反子操纵子。 操纵子中表达的蛋白质之一是蛋白质LepA。 这种蛋白质在翻译过程中起着重要作用，因为它可以将mRNA-tRNA复合物在核糖体中向后移动一步，这有望提高翻译的保真度。-->
 								</p>
 								<p>
-									<span class="subtitle3">6 His-tag: </span>
+									<p class="subtitle">6 His-tag： <!--{cn}6His标签：--></span></p>
-									Currently His-tags have been extensively applied for recombinant protein expression. Based on our literature search, 6His-tag seems to provide enough spacing for protein folding to prevent crystallization or misfolding of peptide chain, and it is a commonly used in protein purification by chromatography.
+									Currently His-tags have been extensively applied for recombinant protein expression. Based on our literature search, 6His-tag seems to provide enough spacing for protein folding to prevent crystallization or misfolding of peptide chain, and it is a commonly used in protein purification by chromatography.<!--{cn}目前，His标签已广泛应用于重组蛋白表达。 根据我们的文献检索，6His标签似乎为蛋白质折叠提供了足够的间隔以防止肽链的结晶或错误折叠，并且它通常通过色谱法用于蛋白质纯化。-->
 								</p>
 								<p>
-									<span class="subtitle3">RBS: </span>
+									<p class="subtitle">RBS： </span></p>
-									The strong ribosome binding site (RBS) is a shine-Dalgarno sequence from gsiB gene. It can lead to a pronounced stimulation of expression when placed upstream of a variety of genes, and significant increase in the translation of the genes is observed.
+									The strong ribosome binding site (RBS) is a shine-Dalgarno sequence from gsiB gene. It can lead to a pronounced stimulation of expression when placed upstream of a variety of genes, and significant increase in the translation of the genes is observed.<!--{cn}强核糖体结合位点（RBS）是来自gsiB基因的shine-Dalgarno序列。 当置于多种基因的上游时，它可以导致表达的显着刺激，并且观察到基因翻译的显着增加。-->
 								</p>
 								<p>
-									<span class="subtitle3">Signal peptide: </span>
+									<p class="subtitle">Signal peptide: <!--{cn}信号肽：--></span></p>
-									Bacillus subtilis is a well-known chassis with highly active protein secretion system, There are mainly two protein export pathways availibale in B.subtilis: Sec-dependent translocation pathway and twin-arginine translocation (Tat) pathway
+									<i>Bacillus subtilis<!--{cn}枯草芽孢杆菌--></i>
+									 is a well-known chassis with highly active protein secretion system, There are mainly two protein export pathways availibale in B.subtilis: Sec-dependent translocation pathway and twin-arginine translocation (Tat) pathway<!--{cn}是一种具有高活性蛋白分泌系统的着名底盘，在枯草芽孢杆菌中主要有两种蛋白质输出途径：Sec依赖易位途径和双精氨酸易位（Tat）途径-->
 									<sup>[14]</sup>
-									. Compare with Sec pathway, tat pathway has an intrinsic advantage of being able to transport folded proteins across the cytoplasmic membrane and without the requirement ATP hydrolysis, this helps retaining protein function extracellularly
+									. Compare with Sec pathway, tat pathway has an intrinsic advantage of being able to transport folded proteins across the cytoplasmic membrane and without the requirement ATP hydrolysis, this helps retaining protein function extracellularly<!--{cn}。 与Sec途径相比，tat途径具有能够将折叠的蛋白质转运穿过细胞质膜并且不需要ATP水解的内在优势，这有助于在细胞外保留蛋白质功能。-->
 									<sup>[15]</sup>
-									. Tat pathway has a signature twin-arginine (RR) motif located at the border of the N-terminal domain and the hydrophobic region. AmyX signal peptide belongings to tat pathway. In our project AmyX was fused in N-terminal of the LHyal gene.
+									. Tat pathway has a signature twin-arginine (RR) motif located at the border of the N-terminal domain and the hydrophobic region. AmyX signal peptide belongings to tat pathway. In our project AmyX was fused in N-terminal of the LHyal gene.<!--{cn}。 Tat途径具有位于N-末端结构域和疏水区域边界的标记双精氨酸（RR）基序。 AmyX信号肽属于tat途径。 在我们的项目中，AmyX融合在LHyal基因的N末端。-->
 								</p>
 								<p>
-									<span class="subtitle3">pDG1730 plasmid backbone </span>
+									<p class="subtitle">pDG1730 plasmid backbone： <!--{cn}pDG1730质粒骨架：--></span></p>
-									contains Bsu-amyE homologous arm sequences that help LHyal construct to be integrated into the amyE locus of B.subtilis genome by double-crossover integration. There is also an ori sequence for replication in E. Coli, three antibiotic resistant genes for E.coli and B. subtilis selections (Amp, Erm and Spc), and a T1 terminator sequence. the whole construct was sub-cloned into the backbone at the BamHI and HindIII restriction sites.
+									contains Bsu-amyE homologous arm sequences that help LHyal construct to be integrated into the amyE locus of <!--{cn}含有Bsu-amyE同源臂序列，通过双交换整合帮助LHyal构建体整合到-->
+									<i>B.subtilis<!--{cn}枯草芽孢杆菌--></i>
+									 genome by double-crossover integration. There is also an ori sequence for replication in <!--{cn}基因组的amyE基因座中。 在-->
+									<i>E. Coli<!--{cn}大肠杆菌--></i>
+									 , three antibiotic resistant genes for <!--{cn}中还存在用于复制的ori序列，用于-->
+									<i>E.coli<!--{cn}大肠杆菌--></i>
+									  and <!--{cn}和-->
+									<i>B. subtilis<!--{cn}枯草芽孢杆菌--></i>
+									  selections (Amp, Erm and Spc), and a T1 terminator sequence. the whole construct was sub-cloned into the backbone at the BamHI and HindIII restriction sites.<!--{cn}选择的三种抗生素抗性基因（Amp，Erm和Spc）和T1终止子序列。 将整个构建体亚克隆到BamHI和HindIII限制性位点的骨架中。-->
 								</p>
 								<p>
-									<span class="subtitle3">pMA0911 plasmid backbone </span>
+									<p class="subtitle">pMA0911 plasmid backbone： <!--{cn}pMA0911质粒骨架：--></span></p>
-									consists of two antibiotic resistant genes which are used for different chassis selections (Kane, Amp) and an E. coli ori sequence. The construct was sub-cloned into the backbone at the NdeI and BamHI restriction sites.
+									consists of two antibiotic resistant genes which are used for different chassis selections (Kane, Amp) and an <!--{cn}由两种抗生素抗性基因组成，用于不同的底盘选择（Kane，Amp）和-->
+									<i>E. coli<!--{cn}大肠杆菌--></i>
+									 ori sequence. The construct was sub-cloned into the backbone at the NdeI and BamHI restriction sites.<!--{cn}ori序列。 将构建体亚克隆到NdeI和BamHI限制性位点的骨架中。-->
 								</p>
 							</div>
@@ Line 376: / Line 402: @@
 							<div class="Three">
 								<p>
-									<span class="subtitle3">LHAase transformation and expression</span>
-									<br />
+									<p class="subtitle">Transformation and Expression<!--{cn}转化和表达--></p>
-									pDG1730-LHyal construct vector was transformed into B.subtilits 168 by chemical transformation method and grew on LB agar+antibiotic at 37℃overnight. Positive selection of integration was performed with spectinomycin at 100μg/ml and negative selection of single crossover integration events with erythromycin at 0.5μg/ml. Colony PCR for verifying part integration were realized using Taq polymerase PCR system (Takara).
+									<p class="subtitle3">① Transformation<!--{cn}①转化--></span></p>
+									pDG1730-LHyal construct vector was transformed into <!--{cn}通过化学转化方法将pDG1730-LHyal构建体载体转化到-->
+									B.subtilits 168<!--{cn}枯草芽孢杆菌168-->
+									 by chemical transformation method and grew on LB agar+antibiotic at 37℃overnight. Positive selection of integration was performed with spectinomycin at 100μg/ml and negative selection of single crossover integration events with erythromycin at 0.5μg/ml. Colony PCR for verifying part integration were realized using Taq polymerase PCR system (Takara).<!--{cn}中，并在LB琼脂+抗生素上于37℃生长过夜。 用100μg/ ml的壮观霉素进行积分的正选择，并用0.5μg/ ml的红霉素进行单交换整合事件的阴性选择。 使用Taq聚合酶PCR系统（Takara）实现用于验证部件整合的菌落PCR。-->
 								</p>
 								<p>
-									The primers for colony PCR polymerization:
+									The primers for colony PCR polymerization: <!--{cn}用于菌落PCR聚合的引物：-->
 									<br />
 									<em>LHAase-F:</em> ATGAAAGAGATCGCGGTGA
@@ Line 388: / Line 417: @@
 								</p>
 								<p>
-									<span class="subtitle3">Fed-batch fermentation</span>
+									<p class="subtitle3">② Fed-batch fermentation<!--{cn}②补料分批发酵--></span></p>
-									<br />
+									Once colonies contain recombinant bacteria were identified, single colony was streaked out into a starter 1ml LB culture (containing 50ug/ml erythromycin) and grew at 30℃, 220rpm, overnight culture was inoculated into 50ml LB medium+ 50ug/ml erythromycin (1% v/v) and grew for 12 h at shaking before fresh 50ml LB medium+ 50ug/ml erythromycin was added. Every 12 h, fresh 50ml LB with antibiotic was fed until 48 h later a 200ml culture was achieved. Cell density was measured every 12 h. After the final feed, when OD600 reached ~2.5, the bacterial cells were removed by centrifugation and filtered through a 0.45um micro-membrane. Culture supernatant was freeze-dried to become powder before further purification.<!--{cn}一旦发现菌落含有重组细菌，将单个菌落划线入1ml LB培养基（含有50ug / ml红霉素）并在30℃，220rpm下生长，过夜培养物接种到50ml LB培养基+ 50ug / ml红霉素（1％v）中 / v）并在摇动前生长12小时，然后加入新鲜的50ml LB培养基+ 50ug / ml红霉素。 每12小时，喂食含有抗生素的新鲜50ml LB直至48小时后，实现200ml培养。 每12小时测量细胞密度。 在最终进料后，当OD600达到~2.5时，通过离心除去细菌细胞并通过0.45um微膜过滤。 在进一步纯化之前，将培养上清液冷冻干燥成粉末。-->
-									Once colonies contain recombinant bacteria were identified, single colony was streaked out into a starter 1ml LB culture (containing 50ug/ml erythromycin) and grew at 30℃, 220rpm, overnight culture was inoculated into 50ml LB medium+ 50ug/ml erythromycin (1% v/v) and grew for 12 h at shaking before fresh 50ml LB medium+ 50ug/ml erythromycin was added. Every 12 h, fresh 50ml LB with antibiotic was fed until 48 h later a 200ml culture was achieved. Cell density was measured every 12 h. After the final feed, when OD600 reached ~2.5, the bacterial cells were removed by centrifugation and filtered through a 0.45um micro-membrane. Culture supernatant was freeze-dried to become powder before further purification.
 								</p>
 								<p>
-									<span class="subtitle3">LHAase purification</span>
+									<p class="subtitle3">③ LHAase purification<!--{cn}③LHAase纯化--></span></p>
-									<br />
+									LHAase was isolated and purified from freeze-dried supernatant powder by Ni-NTA spin columns (Biorad Cat.no. 31314). Briefly, dried powder was dissolved into deionized water and added 5ul RNase then the 630ul enzyme liquid was added to Ni-NTA column [QIAGEN Co Ltd.Cat NO31314] for 5min, the non-target proteins were eluted by using 600ul NPI-20 (50mM NaH2PO4, 300mMNaCl, 20mM imidazole) and was centrifugated 2 times, supernatant was collected by 890g centrifugation. Target protein was then eluted by using 300ul NPI-500 (50mM NaH2PO4, 300mMNaCl, 500mM imidazole) and supernatant collected by centrifugation. Purified enzyme was dialyzed (Regenerated cellulose dialysis membrane MWCO 8000-14000) against 50mM phosphate buffer (pH:6-7.4) overnight before HA was freeze-dried. Purified HA powder was analyzed by SDS-Polyacrylamide gel electrophoresis (PAGE) and enzymatic analyses.<!--{cn}通过Ni-NTA旋转柱（Biorad Cat.no.31314）从冷冻干燥的上清液粉末中分离和纯化LHAase。 简言之，将干燥的粉末溶解在去离子水中并加入5ul RNase，然后将630ul酶液加入Ni-NTA柱[QIAGEN Co Ltd.Cat NO31314]中5分钟，用600ul NPI-20洗脱非靶蛋白（ 将50mM NaH 2 PO 4,300mM NaCl，20mM咪唑）离心2次，通过890g离心收集上清液。 然后通过使用300ul NPI-500（50mM NaH 2 PO 4,300mM NaCl，500mM咪唑）洗脱靶蛋白，并通过离心收集上清液。 在HA冷冻干燥之前，将纯化的酶对50mM磷酸盐缓冲液（pH：6-7.4）透析过夜（再生纤维素透析膜MWCO 8000-14000）。 通过SDS-聚丙烯酰胺凝胶电泳（PAGE）和酶分析分析纯化的HA粉末。-->
-									LHAase was isolated and purified from freeze-dried supernatant powder by Ni-NTA spin columns (Biorad Cat.no. 31314). Briefly, dried powder was dissolved into deionized water and added 5ul RNase then the 630ul enzyme liquid was added to Ni-NTA column [QIAGEN Co Ltd.Cat NO31314] for 5min, the non-target proteins were eluted by using 600ul NPI-20 (50mM NaH2PO4, 300mMNaCl, 20mM imidazole) and was centrifugated 2 times, supernatant was collected by 890g centrifugation. Target protein was then eluted by using 300ul NPI-500 (50mM NaH2PO4, 300mMNaCl, 500mM imidazole) and supernatant collected by centrifugation. Purified enzyme was dialyzed (Regenerated cellulose dialysis membrane MWCO 8000-14000) against 50mM phosphate buffer (pH:6-7.4) overnight before HA was freeze-dried. Purified HA powder was analyzed by SDS-Polyacrylamide gel electrophoresis (PAGE) and enzymatic analyses.
 								</p>
 								<p>
-									<span class="subtitle3">SDS-PAGE</span>
+									<p class="subtitle3">④ SDS-PAGE<!--{cn}④聚丙烯酰胺凝胶电泳--></span></p>
-									<br />
+									Appropriate freeze-dried LHAase powder was mixed with 6xloading buffer (30mM EDTA,36%(v/v) glycerol, 0.05%(w/v), xylene cyanol FF, and 0.05% (w/v) Bromophenol blue). Sample was denatured by boiling for 10 min, and then loaded into the gel. Electrophoresis was performed at 150V, 80mA for 1 h. Gel was stained and destained before photography.<!--{cn}将合适的冷冻干燥的LHAase粉末与6x上样缓冲液（30mM EDTA，36％（v / v）甘油，0.05％（w / v），二甲苯蓝FF和0.05％（w / v）溴酚蓝）混合。 通过煮沸10分钟使样品变性，然后加载到凝胶中。 电泳在150V，80mA下进行1小时。 凝胶在拍照呈现前染色并脱色。-->
-									Appropriate freeze-dried LHAase powder was mixed with 6xloading buffer (30mM EDTA,36%(v/v) glycerol, 0.05%(w/v), xylene cyanol FF, and 0.05% (w/v) Bromophenol blue). Sample was denatured by boiling for 10 min, and then loaded into the gel. Electrophoresis was performed at 150V, 80mA for 1 h. Gel was stained and destained before photography.
 								</p>
 							</div>
@@ Line 407: / Line 433: @@
 							<div class="four">
 								<p>
-									<span class="subtitle3">Agarose Plate Assay by using HA as a substrate</span>
+									<p class="subtitle">Enzymatic Characterization of LHAase<!--{cn}LHAase的酶学表征--></p>
-									<br />
+									<p class="subtitle3">① Agarose Plate Assay by using HA as a substrate<!--{cn}①通过使用HA作为底物进行琼脂糖平板测定--></p>
-									LHAase activity was confirmed using the simple plate assay
+									LHAase activity was confirmed using the simple plate assay<!--{cn}使用简单平板测定确认LHAase活性-->
 									<sup>[16]</sup>
-									. The assay plate was prepared with 1mg/ml HA, 0.75g agarose, 50mM sodium citrate (pH5.5) buffer. 150ul sample, control or standard were injected into the cylindrical holes (3mm in diameter) on the agarose plates, incubated at 37℃ for 10h and covered with 10%(w/v) cetylpyridinium chloride. The formation of a distinct clear halo around the hole indicates HAase activity. Standard enzyme solution (hyaluronidase extracted from bovine testis) was set as positive control, heat-inactivated enzyme solution was set as negative control.
+									. The assay plate was prepared with 1mg/ml HA, 0.75g agarose, 50mM sodium citrate (pH5.5) buffer. 150ul sample, control or standard were injected into the cylindrical holes (3mm in diameter) on the agarose plates, incubated at 37℃ for 10h and covered with 10%(w/v) cetylpyridinium chloride. The formation of a distinct clear halo around the hole indicates HAase activity. Standard enzyme solution (hyaluronidase extracted from bovine testis) was set as positive control, heat-inactivated enzyme solution was set as negative control. <!--{cn}。 用1mg / ml HA，0.75g琼脂糖，50mM柠檬酸钠（pH5.5）缓冲液制备测定板。 将150ul样品，对照或标准品注入琼脂糖平板上的圆柱形孔（直径3mm）中，在37℃下孵育10h并用10％（w / v）氯化十六烷基吡啶覆盖。 在孔周围形成明显的清晰晕圈表明HAase活性。 将标准酶溶液（从牛睾丸中提取的透明质酸酶）设定为阳性对照，将热灭活的酶溶液设定为阴性对照。-->
 								</p>
 								<p>
-									<span class="subtitle3">Enzyme Linked Immunosorbent Assay (ELISA)</span>
+									<p class="subtitle3">② Enzyme Linked Immunosorbent Assay (ELISA)<!--{cn}②酶联免疫吸附试验（ELISA）--></span></p>
-									<br />
+									ELISA is a very sensitive immunochemical technique used to assess the presence of specific protein (antigen or antibody) in the given sample and its quantity. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. LHAase activity was studied using a microorganism HA ELISA kit (Tongwei Ltd, Shanghai, Cat.no tw045410). Firstly, 40μl standard and 10ul sample were mixed and loaded into a 96 well microplate. No sample is added to the blank well. 100μl enzyme-conjugate was added to standard wells and sample wells except the blank well, then cover with an adhesive strip and incubate for 60min at 37℃. The microplate was washed 4 times using 20x eluent. Substrate A (50μl) and Substrate B (50μl) were added to each well. Gently mix and incubate for 15m at 37℃, stop solution was added to each well to induce color changes. The Optical Density (O.D.) is read at 450 nm using a microtiter plate reader within 15min. This assay was performed in duplicates and repeated three times.<!--{cn}ELISA是一种非常灵敏的免疫化学技术，用于评估给定样品中特定蛋白质（抗原或抗体）的存在及其数量。与抗体缀合的酶与无色底物反应，产生有色反应产物。使用微生物HA ELISA试剂盒（Tongwei Ltd，Shanghai，Cat.no tw045410）研究LHAase活性。首先，将40μl标准品和10μl样品混合并装入96孔微量培养板中。没有样品添加到空白孔中。将100μl酶 - 缀合物加入到标准孔和样品孔中，除了空白孔，然后用粘合剂条覆盖并在37℃下孵育60分钟。使用20x洗脱液将微孔板洗涤4次。向每个孔中加入底物A（50μl）和底物B（50μl）。在37℃轻轻混合并孵育15分钟，向每个孔中加入终止溶液以诱导颜色变化。使用微量滴定板读数器在15分钟内在450nm处读取光密度（OD）。该测定一式两份进行并重复三次。-->
-									ELISA is a very sensitive immunochemical technique used to assess the presence of specific protein (antigen or antibody) in the given sample and its quantity. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. LHAase activity was studied using a microorganism HA ELISA kit (Tongwei Ltd, Shanghai, Cat.no tw045410). Firstly, 40μl standard and 10ul sample were mixed and loaded into a 96 well microplate. No sample is added to the blank well. 100μl enzyme-conjugate was added to standard wells and sample wells except the blank well, then cover with an adhesive strip and incubate for 60min at 37℃. The microplate was washed 4 times using 20x eluent. Substrate A (50μl) and Substrate B (50μl) were added to each well. Gently mix and incubate for 15m at 37℃, stop solution was added to each well to induce color changes. The Optical Density (O.D.) is read at 450 nm using a microtiter plate reader within 15min. This assay was performed in duplicates and repeated three times.
 								</p>
 								<p>
-									<span class="subtitle3">DNS reducing sugar method</span>
+									<p class="subtitle3">③ DNS reducing sugar method<!--{cn}③DNS还原糖法--></span></p>
-									<br />
+									LHAase activity analysis was further examined by measuring the amount of reducing sugar liberated from HA with the 3,5-dinitrosalicylic acid (DNS) colorimetric spectrophotometric<!--{cn}通过用3,5-二硝基水杨酸（DNS）比色分光光度法测量从HA中释放的还原糖的量，进一步检测LHAase活性分析-->
-									LHAase activity analysis was further examined by measuring the amount of reducing sugar liberated from HA with the 3,5-dinitrosalicylic acid (DNS) colorimetric spectrophotometric
 									<sup>[17]</sup>
-									. One unit of enzymatic activity was tentatively defined as equal to the reducing power of glucuronic acid (glucose equivalents in micrograms) liberated per hour from HA at 38℃ and pH5.5. Specific activity was defined as units of enzyme per mg of protein.
+									. One unit of enzymatic activity was tentatively defined as equal to the reducing power of glucuronic acid (glucose equivalents in micrograms) liberated per hour from HA at 38℃ and pH5.5. Specific activity was defined as units of enzyme per mg of protein.<!--{cn}。 暂时将一个酶活性单位定义为等于每小时从HA在38℃和pH5.5下释放的葡萄糖醛酸（葡萄糖当量，以微克计）的还原能力。 比活性定义为每mg蛋白质的酶单位。-->
 								</p>
 								<p>
-									In our experiment, the enzymatic reaction contained 1.6 mg/ml of HA as a substrate and an appropriate amount of LHAase in 50 mM pH 5.5 citrate buffer in a total volume of 1 ml, and was incubated at 38℃ for 10 min. The reaction was stopped by immersion in boiling water for 5 min and before adding DNS solution and further boiled for 15min to induce color changes. Standard enzyme solution (hyaluronidase extracted from bovine testis) was set as positive control, heat-inactivated enzyme solution was set as negative control.
+									In our experiment, the enzymatic reaction contained 1.6 mg/ml of HA as a substrate and an appropriate amount of LHAase in 50 mM pH 5.5 citrate buffer in a total volume of 1 ml, and was incubated at 38℃ for 10 min. The reaction was stopped by immersion in boiling water for 5 min and before adding DNS solution and further boiled for 15min to induce color changes. Standard enzyme solution (hyaluronidase extracted from bovine testis) was set as positive control, heat-inactivated enzyme solution was set as negative control.<!--{cn}在我们的实验中，酶促反应含有1.6mg / ml HA作为底物和适量的LHAase在50mM pH 5.5柠檬酸盐缓冲液中，总体积为1ml，并在38℃温育10分钟。 通过浸入沸水中5分钟并在加入DNS溶液之前停止反应并进一步煮沸15分钟以引起颜色变化。 将标准酶溶液（从牛睾丸中提取的透明质酸酶）设定为阳性对照，将热灭活的酶溶液设定为阴性对照。-->
 								</p>
 							</div>
@@ Line 433: / Line 457: @@
 							<div class="five">
 								<p>
-									Currently, low molecular weight HA has attracted considerable attention because of its potential applications attributed to its unique biological properties, including the stimulation of fibroblast proliferation, collagen synthesis, and potential effects in eliminating certain cancer cells
+									<p class="subtitle">(1)LMW-HA Synthesis<!--{cn}（1）LMW-HA合成--></span></p>
+									Currently, low molecular weight HA has attracted considerable attention because of its potential applications attributed to its unique biological properties, including the stimulation of fibroblast proliferation, collagen synthesis, and potential effects in eliminating certain cancer cells<!--{cn}目前，由于其独特的生物学特性，包括刺激成纤维细胞增殖，胶原合成以及消除某些癌细胞的潜在作用，低分子量HA因其潜在应用而备受关注。-->
 									<sup>[18]</sup>
-									. Compare with high-molecular-weight HAs, LMW-HAs can be readily absorbed by human body and contribute to the biosynthesis of HMW-HA
+									. Compare with HMW-HAs, LMW-HAs can be readily absorbed by human body and contribute to the biosynthesis of HMW-HA<!--{cn}。 与HMW-HA相比，LMW-HA易被人体吸收，有助于HMW-HA的生物合成-->
 									<sup>[19]</sup>
-									. With its important values in medical and cosmetics, it is beneficial to directly obtain low-molecular weight HA from microbial fermentation via LHAase hydrolysis.
+									. With its important values in medical and cosmetics, it is beneficial to directly obtain low-molecular weight HA from microbial fermentation via LHAase hydrolysis.<!--{cn}。 由于其在医学和化妆品中的重要价值，通过LHAase水解酶从微生物发酵直接获得低分子量HA是有益的。-->
 								</p>
-								<p>
+								 <p class="subtitle3">① Transformation<!--{cn}①转化--></span></p>
-									<span class="subtitle3">Transformation</span>
+									pDG1730-LHAyal and pMA0911-LHAyal vectors were transformed into the recombinant strain <!--{cn}将pDG1730-LHAyal和pMA0911-LHAyal载体转化到重组菌株中-->
-									<br />
+									<i>B.subtilis 168E<!--{cn}枯草芽孢杆菌168E--></i>
-									pDG1730-LHAyal and pMA0911-LHAyal vectors were transformed into the recombinant strain
+									, respectively, and selected by antibiotic kanamycin to study the direct biosynthesis of LMW-HA in <!--{cn}分别用抗生素卡那霉素选择，研究LMW-HA的直接生物合成-->
-									<i>B.subtilis 168E</i>
+									<i>B.subtilis 168E-LHAase<!--{cn}枯草芽孢杆菌168E-LHAase--></i>
-									, respectively, and selected by antibiotic kanamycin to study the direct biosynthesis of LMW-HA in
+									. Colony PCR and restriction enzyme digestion confirmed that these vectors was successfully transformed into <!--{cn}。 菌落PCR和限制酶消化证实这些载体成功转化为-->
-									<i>B.subtilis</i>
+									<i>B.subtilis 168E-LHAase<!--{cn}枯草芽孢杆菌168E-LHAase--></i>
-									. Colony PCR and restriction enzyme digestion confirmed that these vectors was successfully transformed into
-									<i>B.subtilis</i>
 									.
 								</p>
 								<p>
-									The primers of colony PCR which is used for verification:
+									<p>The primers of colony PCR which is used for verification: <!--{cn}用于验证的菌落PCR引物：--></p>
-									<br />
 									<em>HasA-F:</em> GGTCCATAGGGCTACAAAAG
 									<br />
@@ Line 462: / Line 484: @@
 								</p>
 								<p>
-									<span class="subtitle3">Fermentation and HA purification</span>
+									<p class="subtitle3">② Fermentation and HA purification<!--{cn}②发酵和HA纯化--></span></p>
-									<br />
+									Refer to HA Fed-batch fermentation of HA and HA separation and purification<!--{cn}参考HA和HA分离和纯化的HA补料分批发酵-->
-									Refer to HA Fed-batch fermentation of HA and HA separation and purification
 								</p>
 								<p>
-									<span class="subtitle3">HA characterization</span>
+									<p class="subtitle">(2)HA characterization<!--{cn}（2）HA表征--></span></p>
-									<br />
+									<p class="subtitle3">① CTAB</span></p>
-									<span class="subtitle3">CTAB: </span>
+									Refer to CTAB (cetyltrimethylammonium bromide) method<!--{cn}参考CTAB（十六烷基三甲基溴化铵）法-->
-									<br />
-									Refer to CTAB (cetyltrimethylammonium bromide) method
 								</p>
 								<p>
-									<span class="subtitle3">HA molecular weight easurement--GPC-RI-MALS</span>
+									<p class="subtitle3">② HA molecular weight easurement--GPC-RI-MALS<!--{cn}④HA分子量测量 - GPC-RI-MALS--></span></p>
-									<br />
+									Gel chromatography (GPC-RI-MALS) is an effective determination of molecular weight of polysaccharides, especially for low-molecular-weight HA that cannot be directly measured using a viscometer<!--{cn}凝胶色谱（GPC-RI-MALS）是一种有效测定多糖分子量的方法，尤其适用于不能用粘度计直接测量的低分子量HA-->
-									Gel chromatography (GPC-RI-MALS) is an effective determination of molecular weight of polysaccharides, especially for low-molecular-weight HA that cannot be directly measured using a viscometer
 									<sup>[20]</sup>
-									. The principle is: when samples of different molecular weights pass through gel column, the distance and the time of the process will be changed according to different molecular weight of HA, therefore different substances can be separated. Then the molecular weight and abundance of elements can be detected by differential detector and multiple angle laser light scattering.
+									. The principle is: when samples of different molecular weights pass through gel column, the distance and the time of the process will be changed according to different molecular weight of HA, therefore different substances can be separated. Then the molecular weight and abundance of elements can be detected by differential detector and multiple angle laser light scattering.<!--{cn}。 原理是：当不同分子量的样品通过凝胶柱时，工艺的距离和时间将根据HA的不同分子量而改变，因此可以分离出不同的物质。 然后通过差分检测器和多角度激光散射检测元素的分子量和丰度 -->
 								</p>
 								<p>
-									GPC-RI-MALS service was provided by Sanshu Biotechnology Co, Shanghai. Purified HA samples were sent to them. To test our HA samples, the mobile phase of the measurement is NaNO3, 10mg of HA sample was dissolved respectively by adding 1.5ml NaNO3, then the mixtures were centrifuged (12000rpm/10min) and filtered through a 0.22um filter membrane. Ultimately 100ul sample was analyzed by gel chromatography.
+									GPC-RI-MALS service was provided by Sanshu Biotechnology Co, Shanghai. Purified HA samples were sent to them. To test our HA samples, the mobile phase of the measurement is NaNO3, 10mg of HA sample was dissolved respectively by adding 1.5ml NaNO3, then the mixtures were centrifuged (12000rpm/10min) and filtered through a 0.22um filter membrane. Ultimately 100ul sample was analyzed by gel chromatography.<!--{cn}GPC-RI-MALS服务由上海三黍生物科技有限公司提供。 将纯化的HA样品送至它们。 为了测试我们的HA样品，测量的流动相是NaNO 3，通过添加1.5ml NaNO 3分别溶解10mg HA样品，然后将混合物离心（12000rpm / 10min）并通过0.22um滤膜过滤。 最后通过凝胶色谱分析100ul样品。-->
 								</p>
 							</div>
@@ Line 490: / Line 508: @@
 				</section>
-				<!--Model 3 HA Micro-needle Design-->
+				<!--Module 3 HA Micro-needle Design-->
 				<section class="col-xs-12 col-sm-10">
-					<p class="title">Module 3 - HA Micro-needle Design</p>
+					<p class="title">Module 3- HA Micro-needle Design<!--{cn}第3模块 - HA微针的设计--></p>
 					<div class="content">
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span class="active">Micro-needle Design</span>
+							<span class="active">1. Micro-needle Concept<!--{cn}1.微针概念--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>Fabrication of cHA Hydrogel Particles</span>
+							<span>2. Fabrication of cHA Hydrogel Particles<!--{cn}2.cHA水凝胶颗粒的制备--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>Preparation of The Micro-needle Mold</span>
+							<span>3. Preparation of the Micro-needle Mold<!--{cn}3.微针模具的制备--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>Fabrication of HA-cHA-micro-needles</span>
+							<span>4. Fabrication of HA-cHA-Micro-needles<!--{cn}4.HA-cHA-微针的制造--></span>
 						</div>
 						<div class="Csubtitle col-xs-12 col-sm-6">
-							<span>Water Solubility Measurement</span>
+							<span>5. Water Solubility Test<!--{cn}5.水溶解度测量--></span>
 						</div>
 						<div class="clearfix"></div>
@@ Line 514: / Line 532: @@
 							<!--One-->
 							<div class="One">
-								<p>
+									<p class="subtitle">Micro-needle Design<!--{cn}透明质酸微针设计--></span></p>
-									At present, the preparation of micro-needles with natural and degradable polymer bio-materials has become a hot topic. In this project we designed a new type of micro-needles by using HA products from previous section.
+									At present, the preparation of micro-needles with natural and degradable polymer bio-materials has become a hot topic. In this project we designed a new type of micro-needles by using HA products from previous section.<!--{cn}目前，用天然可降解聚合物生物材料制备微针已成为热门话题。 在这个项目中，我们使用前一部分的HA产品设计了一种新型微针。-->
 								</p>
-								<p>
+									Cross-link is widely used in dermal filler preparation by linking one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers<!--{cn}交联通过将一条聚合物链连接到另一条聚合物链而广泛用于皮肤填充剂制备中。 这些连接可以采用共价键或离子键的形式，聚合物可以是合成聚合物或天然聚合物-->
-									Cross-link is widely used in dermal filler preparation by linking one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers
 									<sup>[21]</sup>
-									. In polymer chemistry, "cross-linking" usually refers to the use of cross-linking agents to promote a change in the polymers' physical properties.
+									. In polymer chemistry, "cross-linking" usually refers to the use of cross-linking agents to promote a change in the polymers' physical properties.<!--{cn}。 在聚合物化学中，“交联”通常是指使用交联剂来促进聚合物物理性质的变化-->。
 								</p>
 								<p>
-									At present, the mainstream chemical cross-linking agents for cosmetic HA implant preparations are diethylsulfone (DVS) and 1, 4-butanediol diglycidyl ether (BDDE)
+									At present, the mainstream chemical cross-linking agents for cosmetic HA implant preparations are diethylsulfone (DVS) and 1, 4-butanediol diglycidyl ether (BDDE)<!--{cn}目前，用于化妆品HA植入制剂的主流化学交联剂是二乙基砜（DVS）和1,4-丁二醇二缩水甘油醚（BDDE）。-->
 									<sup>[22]</sup>
-									. DVS, although widely used in R&D, has high cytotoxicity. Not only this toxic substance is likely to be accumulated in the body after implantation, but also it may affect normal tissue growth due to calcification of the implant. In comparison, BDDE is the most commonly used cross-linking agent, it is biodegradable, much less toxic and more reactive than DVS, thereby safer for biomedical applications. Cross-linked with HA using BDDE making it more stable, less susceptible to enzymatic degradation, and with increased mechanical strength
+									. DVS, although widely used in R&D, has high cytotoxicity. Not only this toxic substance is likely to be accumulated in the body after implantation, but also it may affect normal tissue growth due to calcification of the implant. In comparison, BDDE is the most commonly used cross-linking agent, it is biodegradable, much less toxic and more reactive than DVS, thereby safer for biomedical applications. Cross-linked with HA using BDDE making it more stable, less susceptible to enzymatic degradation, and with increased mechanical strength<!--{cn}。 DVS虽然广泛用于研发，但具有高细胞毒性。 植入后不仅这种有毒物质可能在体内积聚，而且由于植入物的钙化，它也可能影响正常的组织生长。 相比之下，BDDE是最常用的交联剂，它是可生物降解的，毒性低且比DVS更具反应性，因此对生物医学应用更安全。 使用BDDE与HA交联，使其更稳定，不易受酶降解，并具有增加的机械强度-->
 									<sup>[23]</sup>
-									. Previous studies
+									. Previous studies<!--{cn}。 之前的了解-->
 									<sup>[24]</sup>
-									 showed that under alkaline condition, the opening of BDDE rings during reaction reacts with the OH group of HA to produce crosslinked network (hydrogel).
+									 showed that under alkaline condition, the opening of BDDE rings during reaction reacts with the OH group of HA to produce crosslinked network (hydrogel).<!--{cn}结果表明，在碱性条件下，反应过程中BDDE环的开环与HA的OH基反应生成交联网络（水凝胶）。-->
 								</p>
 								<p>
-									Micro-needles made with cross-linked HA has better swelling ability and slow bio-degradation that could result to a prolonged effectiveness of the dermal filler, which has the potential to replace surgical approach or injection of HA for anti-wrinkle treatment. In addition, we performed water solubility test in water to understand whether cross-linking, solidifying and curing process could affect HA solubility.
+									Micro-needles made with cross-linked HA has better swelling ability and slow bio-degradation that could result to a prolonged effectiveness of the dermal filler, which has the potential to replace surgical approach or injection of HA for anti-wrinkle treatment. In addition, we performed water solubility test in water to understand whether cross-linking, solidifying and curing process could affect HA solubility.<!--{cn}用交联HA制成的微针具有更好的溶胀能力和缓慢的生物降解，这可以导致真皮填充剂的延长效果，其有可能取代手术方法或注射HA用于抗皱治疗。 此外，我们在水中进行了水溶性测试，以了解交联，固化和固化过程是否会影响HA的溶解度。-->
 								</p>
 								<div class="Cimg col-xs-12 col-sm-6">
-									<img src="https://static.igem.org/mediawiki/2018/a/ad/T--SSTi-SZGD--Design_threeinfo_one.png"/>
+									<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SSTi-SZGD--Design_twoinfo_two.png"/>
 									<p>
-										Fig.9. Hyaluronic acid cross-linking
+										Fig9: Hyaluronic acid cross-linking<!--{cn}图9: 透明质酸交联-->
 									</p>
 								</div>
@@ Line 544: / Line 562: @@
 							<!--Two-->
 							<div class="Two">
-								<p>
+                                  <p class="subtitle">Fabrication of cHA Hydro-gel Particles<!--{cn}cHA水凝胶颗粒的制备--></span></p>
-									Cross-linked HA (cHA) reagent solution was prepared by mixing 200μL of 1,4-butanediol diglycidyl ether (BDDE) into 9.8mL of 0.25mol/L NaOH (pH13). Approximately 1.0g of HA powder was added to the cHA reagent solution and stirred well, and thoroughly mixed at 40°C for 5 hours. The prepared hydrogel was grounded, squeezed out and screened with a 170 mesh sieve to get particles having a diameter of less than 90μm
+									Cross-linked HA (cHA) reagent solution was prepared by mixing 200μL of 1,4-butanediol diglycidyl ether (BDDE) into 9.8mL of 0.25mol/L NaOH (pH13). Approximately 1.0g of HA powder was added to the cHA reagent solution and stirred well, and thoroughly mixed at 40°C for 5 hours. The prepared hydrogel was grounded, squeezed out and screened with a 170 mesh sieve to get particles having a diameter of less than 90μm<!--{cn}通过将200μL的1,4-丁二醇二缩水甘油醚（BDDE）混合到9.8mL的0.25mol / L NaOH（pH13）中来制备交联的HA（cHA）试剂溶液。 将约1.0g HA粉末加入到cHA试剂溶液中并充分搅拌，并在40℃下充分混合5小时。 将制备的水凝胶研磨，挤出并用170目筛筛分，得到直径小于90μm的颗粒-->
 									<sup>[25]</sup>
 									.
 								</p>
+								<div class="Cimg col-xs-12 col-sm-6">
+									<img src="https://static.igem.org/mediawiki/2018/e/e2/T--SSTi-SZGD--Design_threeinfo_two.jpeg"/>
+								</div>
 							</div>
 							<!--Three-->
 							<div class="Three">
-								<p>
+								<p class="subtitle">Preparation of The Micro-needle Mold<!--{cn}微针模具的制备--></span></p>
-									The micro-molds made of resin were prepared by 3D-printed technology provided by the Engineering Department of our school. The needle cavities are cone shape, with 6500um in depth and 2500um in diameter, and on the micro-molds are patterned into 3 × 3 (round shape) and 10 × 10 (rectangular shape).
+									The micro-molds made of resin were prepared by 3D-printed technology provided by the Engineering Department of our school. The needle cavities are cone shape, with 6500um in depth and 2500um in diameter, and on the micro-molds are patterned into 3 × 3 (round shape) and 10 × 10 (rectangular shape).<!--{cn}由树脂制成的微型模具由我校工程部提供的3D打印技术制备。 针腔为锥形，深度为6500um，直径为2500um，在微型模具上图案化为3×3（圆形）和10×10（矩形）。-->
 								</p>
 								<div class="img colxs12">
@@ Line 565: / Line 586: @@
 									<div class="clearfix"></div>
 									<p>
-										Fig：Illustration of micro-needle molds
+										Fig10：Illustration of micro-needle molds<!--{cn}图10：微针模具的图示-->
 									</p>
 								</div>
@@ Line 572: / Line 593: @@
 							<!--Four-->
 							<div class="four">
-								<p>
+								<p class="subtitle">Fabrication of HA-cHA-micro-needles<!--{cn}HA-cHA-微针的制造--></span></p>
-									The HA-cHA-micro-needles was prepared by mixing different proportions of uncross-linked HA powder and cross-linked HA (cHA) powder to become mixtures, the ratios tested were 1:0, 1:1, and 5:1 (HA-cHA)
+									The HA-cHA-micro-needles was prepared by mixing different proportions of uncross-linked HA powder and cross-linked HA (cHA) powder to become mixtures, the ratios tested were 1:0, 1:1, and 5:1 (HA-cHA)<!--{cn}通过将不同比例的未交联的HA粉末和交联的HA（cHA）粉末混合成混合物来制备HA-cHA-微针，测试的比例为1：0,1：1和5：1（HA-CHA）-->
 									<sup>[26]</sup>
-									. The mixtures were dissolved in ultrapure water and a 20% (w/v) viscous polymer solution was stirred at room temperature under a magnetic stirrer for the production of homogenous suspension. The homogenous suspension was poured into the mold, and centrifuged at 10,000 rpm for 10 min to ensure the full loading to the needle cavities. Excess liquid was removed by using a tape. This step was repeated twice until the needle cavity was full, and then the viscous polymer solution was poured onto the surface of the mold, any residual solution remaining at the edge of the mold was removed by a blade. After curing overnight at room temperature, micro-needle was successfully prepared by separating from the mold using a blade.
+									. The mixtures were dissolved in ultrapure water and a 20% (w/v) viscous polymer solution was stirred at room temperature under a magnetic stirrer for the production of homogenous suspension. The homogenous suspension was poured into the mold, and centrifuged at 10,000 rpm for 10 min to ensure the full loading to the needle cavities. Excess liquid was removed by using a tape. This step was repeated twice until the needle cavity was full, and then the viscous polymer solution was poured onto the surface of the mold, any residual solution remaining at the edge of the mold was removed by a blade. After curing overnight at room temperature, micro-needle was successfully prepared by separating from the mold using a blade.<!--{cn}。 将混合物溶解在超纯水中，并在室温下在磁力搅拌器下搅拌20％（w / v）粘性聚合物溶液，以产生均匀的悬浮液。 将均匀的悬浮液倒入模具中，并以10,000rpm离心10分钟以确保完全加载到针腔中。 用胶带除去多余的液体。 将该步骤重复两次直至针腔充满，然后将粘性聚合物溶液倒在模具表面上，用刀片除去残留在模具边缘的任何残余溶液。 在室温下固化过夜后，通过使用刀片从模具中分离成功地制备了微针。-->
 								</p>
 								<div class="Cimg col-xs-12">
 									<img src="https://static.igem.org/mediawiki/2018/a/a2/T--SSTi-SZGD--Design_threeinfo_four.jpeg"/>
 									<p>
-										Fig.10 Flow chart of preparation of microneedle
+										Fig11: Flow chart of preparation of microneedle<!--{cn}图11: 微针制备流程图-->
 									</p>
 								</div>
@@ Line 587: / Line 608: @@
 							<!--Five-->
 							<div class="five">
-								<p>
+								<p class="subtitle">Water Solubility Measurement<!--{cn}水溶性检测--></span></p>
-									Cured micro-needles from 4 were immersed into water at room temperature for 10 min to study water solubility property. Dissolving status was recorded.
+									Cured micro-needles into water at room temperature for 10 min to study water solubility property. Dissolving status was recorded.<!--{cn}将固化微针在室温下浸入水中10分钟以研究水溶性。 记录溶解状况。-->
 								</p>
+								<div class="clearfix"></div>
 							</div>