Revision as of 14:08, 17 October 2018

Team:ZJUT-China - 2018.igem.org

Loading menubar.....

Team:ZJUT-China

Overview

The antimicrobial resistance is mostly caused by the expression of related antibiotic resistance genes(ARGs), which are widely being used as screening markers. But no effective method has been applied to ensure that ARGs would not be released into the environment yet. This situation poses a great threat to the society and is exactly the problem we want to solve.
The goal of our project is to eliminate the ARGs carried by the plasmids of the bacteria themselves. The way of elimination is to cleave the target ARGs by the light-controlled expression of the Cas9 protein through the guidance of sgRNA. So we designed the following gene circuits:

Our goal is to eliminate the ARGs carried on the plasmids used in laboratories and fermentation plants. But it is difficult to build our system on this plasmid because our system is too huge. So weould need a second plasmid as a vector for the light-controlled cleavage system. In this case we plan to move the different ARGs on the different two plasmids, and achieve their occurrence in a certain order. To this end, we have improved the original design:

The sgRNA1 transcribed by the guide1 will guide Cas9 to cleave ARG1 on the plasmid1, and the sgRNA2 transcribed by the guide2 will guide Cas9 to cleave ARG2 on Plasmid 2. When ARG1 on Plasmid 1 is not cleaved, the plasmid is intact, and the normally expressed Repressor will repress the transcription of the guide2. when ARG1 is cleaved, Repressor cannot be expressed normally, resulting in the transcription of the guide2. This leads to ARG2 degradation by Cas9 on the plasmid2. Our design allows the two plasmids to be cleaved with a certain order to ensure self-destruction after elimination of the target gene.

What’s more, in our final design, cells end up with a inducible-lysis after ARGs were cut. To achieve this we designed the following gene circuit for lysis part and it will be added to the genome using CRISPR/Cas9 genome editing tool.

Light-controlled system

1.Primary light control system

1.1.Introduction of primary light control system

The primary light control system is shown in figure 1. Under dark condition, the phosphate group is transferred from Yf1 protein to FixJ protein, the phosphorylated FixJ protein activates the P_fixK2 promoter and then activates the downstream gene expression. When induced by light, the phosphorylation of Fixj protein will be blocked, and the expression of genes regulated by P_fixK2 promoter will be inhibited.

Figure 1. primary light control system

1.2.Experimental design

To further verify the effectiveness of the system, we plan to transfer plasmids (dusk-eGFP-pUC57) with the system to different hosts. Cultured under blue and dark conditions after a period of time, then detected the fluorescence value of eGFP. The efficiency of primary light control system in different hosts was compared.

1.3.Experimental method

1.3.1.Extraction of dusk-eGFP-pUC57 plasmid

Because the 2017 ZJUT team had already constructed the dusk-eGFP-pUC57 plasmid,we used a shaking flask containing 50ml LB liquid medium to incubate bacteria carrying the plsmid overnight, and then extracted the plasmids using the DNA extraction kit. See the specification of the kit for specific operation.

1.3.2.Preparation of competent cells

After discussion, we decided to select DH5α, BL21, MG1655 and BW25113 as the hosts of the primary light control system. When the host cells were cultured to a OD₆₀₀ value of about 0.5, the effect of preparing the competent cells was the best.

1.3.3.Transformation

The transformation was carried out at 42℃. The transformation solution was coated on the LB solid medium containing Amp and cultured overnight at 37℃. Observe the number of transformants.

1.3.4.Primary light control system effect detection

The successful transformants and their corresponding hosts without primary light control system were cultured under blue light and dark light respectively. The expression of eGFP was detected every other time.

2.Improvement of primary light control system

After being cultured at 37℃ for 24 to 48 hours in darkness, the expression of eGFP protein is not very high, showing our light control system lacks efficiency. So we wanted to improve the light control system and had a discussion with the team members. We decided to replace the J23100 promoter with the T7 promoter.

2.1.Replacement of a strong promoter

We planned to replace the J23100 promoter with the T7 promoter to reconstruct the light control system (Fig.3), and determine the relationship between the culture time and the expression of eGFP protein. Different from the primary light control system, the T7 promoter of the secondary light controlled system is regulated by lactose analogues. We planed to use IPTG to regulate the T7 promoter to regulate the efficiency of the light control system.

Figure 3. secondary light controlled system

2.2.Experimental design

We plan to obtain the T7 promoter from the pET28b plasmid, delete the PJ23100 promoter from the dusk-eGFP-pUC57 plasmid, construct the T7-dusk-eGFP-pUC57 plasmid by one step clone method, transfer it into the BL21 host and induce it by adding different amount of IPTG. The expression of eGFP was detected by culturing under blue light and dark light for a period of time.

2.3.Experimental method

2.3.1.Acquisition of T7 Promoter

We obtained the T7 promoter from the pET28b plasmid by PCR. The primers and PCR program are as follows:
pET28b-F：5’-ttgtactgagagtgcaccattaagtgcggcgacgatag-3’
pET28b-R：5’-agtagctagcactgtacctggaattgttatccgctcac-3’

PCR	pET28b
2×Phanta Max Master Mix	25μl
10mM pET28b-F	2μl
10mM pET28b-R	2μl
pET28b plasmid DNA	20~30ng
ddH₂O	to 50μl

PCR condition setting	pET28b
Step one	95℃ 180s
Step two	95℃ 15s	25 cycles
Step three	59℃ 15s	25 cycles
tep four	72℃ 180s	25 cycles
Step five	72℃ 300s

PCR results were detected by electrophoresis gel.

2.3.2.Delete J23100 promoter from dusk-eGFP-pUC57 plasmid

We deleted the J23100 promoter from the dusk-eGFP-pUC57 plasmid by PCR. The primers and the PCR program were designed as follows:
pUC57-F：5’-tgtgagcggataacaattccaggtacagtgctagctact-3’
pUC57-R：5’-gactatcgtcgccgcacttaatggtgcactctcagtac-3’

PCR	dusk-eGFP-pUC57
2×Phanta Max Master Mix	25μl
10mM pUC57-F	2μl
10mM pUC57-R	2μl
pUC57-dusk-eGFP plasmid DNA	20~30ng
ddH₂O	to 50μl

PCR condition setting	pET28b
Step one	95℃ 180s
Step two	95℃ 15s	25 cycles
Step three	59℃ 15s	25 cycles
tep four	72℃ 360s	25 cycles
Step five	72℃ 300s

PCR results were detected by electrophoresis gel.

2.3.3.Template elimination

PCR products need to be treated with Dpn Ⅰ enzyme to remove template plasmids on subsequent experiments. The reaction system is as follows:

Result of template elimination were detected by electrophoresis gel.

2.3.3.One-step cloning and transformation

The insertion fragments and linearized carriers were purified by purification kit, then connected by one-step cloning kit, and then transferred into BL21 competent cells for overnight culture at 37 ℃. The number of transformants were counted. The reaction system of one-step cloning is as follows:

2.3.4.Secondary light control system effect detection

The successful transformants were added to different amounts of IPTG, and cultured under blue light and dark conditions for 24 hours to detect the expression of eGFP.

Constructing a plasmid expressing sgRNA which target chloramphenicol

resisitance gene

▶First step: Test CRISPR/Cas9 system

Overview

We constructed a CRISPR/Cas9 system for degrading ARGs. In this system, plasmid pTargetF expresses sgRNA, to guide Cas9 protein to cleave target gene. Firstly, We tested the CRISPR/Cas9 system. We selected two different kinds of E. coli.The one is wild type, the other is a panD mutant. We transferred pCas plasmid and pTargetF plasmid into the two strains. We observed that the wild type died and the panD mutant grew. The results proved that the CRISPR/Cas9 system was effective.

Functional description

The plasmid pTargetF-panD expresses sgRNA which targets on the panD, which guides Cas9 to the panD gene and cause double-strand DNA break in the genome of E. coli MG1655.

Functional verification

The strain we used is E. coli MG1655 ΔpanD and E. coli MG1655 wild type. Firstly, we need to prepare E. coli MG1655 ΔpanD and E. coli MG1655 wild type competent cells and transform pCas plasmid into it. Then, we need to transform pTargetF-panD plasmid into the above transformants. Finally, the plates were incubated at 30℃ overnight.

pTargetF-panD can guide Cas9 to the panD gene and cause double-strand DNA break in the genome of E. coli MG1655 wide type, while pTargetF-panD can not guide Cas9 to any place in the genome of E. coli MG1655 ΔpanD. Transformants of E. coli MG1655 ΔpanD grew, Transformants of E. coli MG1655 wild type died.

▶Second step: Construct pTargetF-cm

Overview

In order to achieve degradation of ARGs, we have been trying to construct a resistant cutting system by using CRISPR/Cas9 system. In this system, plasmid pTargetF expresses sgRNA, to guide Cas9 protein to the target gene. Secondly, We have been construct pTargetF-cm, which can express sgRNA which targets chloramphenicol resistance gene. And sequencing results showed successful construction.

Functional description

The plasmid pTargetF-cm can expresses sgRNA which targets on the chloramphenicol resistance gene.

Functional verification

After PCR, we got the plasmid pTargetF-cm, then transformed it into E. coli DH5α competent cells.The construction was confirmed by sequancing.

▶Third step: Test pTargetF-cm

Overview

In order to achieve degradation of ARGs, we have been trying to construct a resistant cutting system by using CRISPR/Cas9 system. In this system, plasmid pTargetF expresses sgRNA, for guiding Cas9 protein to cleavage target gene. Thirdly, we tested the function of the plasmid pTargetF-cm.We introduced pSU20 which has a chloramphenicol resistance gene and pCas which has a kanamycin resistance gene into E. coli MG1655. Finally, the transformant can grow in LB supplemented with kanamycin and spectinomycin, while it can not grow in LB supplemented with chloramphenicol. The result showed that pTargetF-cm worked successfully.

Functional description

The plasmid pTargetF-cm expresses sgRNA which targets on the chloramphenicol in E. coli MG1655-pSU20. Therefore, pTargetF-cm can guide Cas9 to the chloramphenicol resistance gene and cause double-strand DNA break in the pSU20 plasmid.

Functional verification

The strain we used is E. coli MG1655 wild type. First, we need to prepare E. coli MG1655 wild type competent cells and transform pSU20 plasmid into it. Then, we need to culture the transformants and prepare them into competent cells, and then transform pCas plasmid. Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-cm plasmid, respectively.

pTargetF-cm can guide Cas9 to the chloramphenicol resistance gene and cause double-strand DNA break in the pSU20 plasmid. After grown at 30℃ overnight,After incubation at 30 C overnight, E. coli MG1655 wild type was able to grow in LB supplemented with kanamycin and spectinomycin but failed to grow in the culture supplemented with chloramphenicol because the corresponding resistance gene in pSU20 had been eliminated.

P_BAD-Cas9

Before we used the light-controlled system to regulate the expression of Cas9, we first used P_BAD to regulate the expression of Cas9 to verify the cleavage function of Cas9.

The arabinose-induced promoter offers the possibility to regulate the expression of Cas9 protein in E. coli by adding or not adding a certain amount of arabinose into the culture.

Experiment design:

We built this part on pGLO and named it pGLO-Cas9. To verify whether the expressed Cas9 protein has the function of cutting the target gene, we used E. coli MG1655 as a chassis. In addition, we will use the pTargetF plasmid to transcribe the sgRNA that targets the panD gene. Since the original pTargetF is not compatible with pGLO, we have built a pTargetF-p15A that is compatible with pGLO. Finally, we characterized the function of the Cas9 gene based on the growth curve of the bacteria.
When the function of Cas9 was verified, we replaced the sgRNA targeting panD with the sgRNA targeting the chloramphenicol gene. The plasmid we constructed was named pTargetF-p15A-cm. And we used E. coli MG1655 containing the chloramphenicol gene on genome for validation experiments.

The experiment is divide into 5 steps:

the regulation of an inducible promoter. This is based on a given plasmid pTargetF, we changed its replicon to make it compatible with P_BAD-Cas9.
1) Construct the P_BAD-Cas9 plasmid based on a given plasmid pGLO.
2) Construct a plasmid pTargetF-p15A to express sgRNA to test if the Cas9 protein is function under the regulation of an inducible promoter. This is based on a given plasmid pTargetF, we changed its replicon to make it compatible with P_BAD-Cas9.
3) Transform both plasmid to E. coli MG1655
4) Get the growth curve result to prove after adding arabinose, the Cas9 protein will express and perform its function.
5) Construct and test a plasmid pTargetF-p15A-cm to perform ARGs cut.
*panD gene is a gene on the genome of E. coli, in the previous research of our lab, we obtained a panD-deficient strain of E. coli.

Light-controlled Cas9 system

1.1Functional description

Under the dark condition, YF1 phosphorylates FixJ and phospholylated FixJ activates the P_fixK2 promoter, which activates the expression of Cas9.Under the blue light condition YF1 don’t phosphorylate FixJ. Therefore P_fixK2 promoter can not initiate the expression of Cas9.

The plasmid pTargetF-cm expresses sgRNA which targets on the chloramphenicol resistance gene . In a panD mutant E. coli Bl21+ΔpanD, the original panD gene was replaced by the chloramphenicol resistance gene in the genome. Therefore, pTargetF-cm can guide Cas9 to the chloramphenicol gene and cause double-strand DNA break in the genome of E. coli Bl21+ΔpanD.

Under dark conditions, cas9 gene can be expressed, so pTargetF-cm can guide Cas9 to the chloramphenicol resistance gene and cause double-strand DNA break in the genome.However, in blue light,the expression of Cas9 is inhibited, double-strand DNA will not be cut.

Figure 1. Light-controlled CRISPR/Cas9 system

1.2Experimental design

1.2.1Design of plasmid needed

In this experiment, we need dusk-Cas9-pUC57 plasmid,pTargetF-cm plasmid, pSU20 plasmid and pTargetF-panD plasmid. We used dusk-eGFP-pUC57 plasmid and pTargetF-panD plasmid to construct dusk-Cas9-pUC57 plasmid and pTargetF-cm plasmid. For detailed experimental procedure, please refer to our notebook. 7.14 7.15 7.16

1.2.2Functional verification

The strain we used is E. coli BL21 ΔpanD, which is a panD mutant, the original panD gene was replaced by the chloramphenicol resistance gene in the genome(cm). First, we need to prepare E. coli BL21 ΔpanD competent cells and transform dusk-Cas9-pUC57 plasmid into it.Then, we transformed pTargetF-cm plasmid, pSU20 plasmid (as control) and pTargetF-panD plasmid (as control) into E. coli BL21 ΔpanD dusk-Cas9-pUC57 separately. The transformants were incubated overnight under dark or blue light conditions.

Under dark conditions, pTargetF-cm can guide Cas9 to the chloramphenicol gene and cause double-strand DNA break in the genome of E. coli Bl21 ΔpanD, while pTargetF-panD can not guide Cas9 to any place in the genome of E. coli Bl21 ΔpanD. The damage to the bacterial genome due to CRISPR/Cas9 can be determined by transformation efficiency. When light was provided, the light-controlled gene expression system was suppressed and therefore the transformation efficiency was higher.

Table 1.Transformation experiments in each group

Repression system

▶First step

Overview

In order to achieve the sequential degradation of ARGs, we have been trying to construct a repression system. In this system, the arabinose-controlled repressor protein was utilized to regulate the transcription of sgRNA. We chose two types of inhibitor-promoter: lacI-P_lac, cI-P_R to test just in case. The LacI repressor is a DNA-binding protein which can bind to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG). The P_R promoter has two DNA binding sites for CI repressor. And transcription would be repressed when CI protein binds to the sites. After experiment, we selected the lacI-P_lac to make up the repression system due to the results.

Design

(1)lacI-P_lac

The genetic circuit is shown in Fig.1.

Fig.1 The genetic circuit of lacI-P_lac

This part is mainly composed of three elements:

①Arabinose induced promoter P_BAD to express the gene of downstream. This part contains the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction.By binding to L (+)-arabinose, AraC changes its conformation and induce the transcription of downstream genes.

②This part contains the lac operator as well as the coding sequence for the repressor lacI. The lacI repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl -beta -D-thiogalactopyranoside (IPTG).

③GFP, as the report gene, can show the degree of repression by repressor.

（2）cI-P_R

The genetic circuit is shown in Fig.2.

Fig.2 The genetic circuit of cI-PR

①Arabinose induced promoter P_BAD to express the gene of downstream.

②This part contains the P_R promoter as well as the coding sequence for the repressor cI. The cI repressor binds to the operator to inhibit transcription in E. coli.

③GFP, as the report gene, can verify the degree of repression by repressor cI .

Description

Different concentrations of arabinose will induce these parts with different concentration of repressor proteins. By adding different concentration of arabinose, we can measure the fluorescence intensity of GFP and find out the relationship between the concentration of arabinose and RFU (Relative fluorescence unit) intensity. And by adding IPTG，the LacI protein is able to be released from the lac operator .It is a characteristic that can be utilized to further verify the function of the lacI-P_lac by adding IPTG or not.

▶Second step

Overview

Based on the results we have achieved, we chose lacI-P_lac to carry out the next step. In this system, the arabinose-controlled repressor protein is utilized to regulate the transcription of sgRNA.

Single-guide RNA (sgRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It contains the 20-bp complementary region (N20) with the requisite NGG PAM matching genomic loci of interest and the sequence of tracrRNA. It will guide Cas9 to the target. We design the sgRNA which specifically targets the gene panD on the E. coli genome in order to demonstrate our CRISPR/Cas9 system.

●lacI-P_lac-sgRNA

The genetic circuit is shown in Fig.3.

Fig.3 The genetic circuit of lacI-Plac-sgRNA

We combined lacI repression system (BBa_K2556031) which is controlled by the AraC-Ara promoter (BBa_K808000) with sgRNA (BBa_K2556041) so that we can regular the transcription of sgRNA. It’s important to activate the CRISPR/Cas9 system only under certain proper conditions.

Description

When activating the AraC-Ara promoter by adding L (+)-arabinose, the LacI repressor will express and the transcription of sgRNA will be inhibited. But this inhibition can be relieved after induction with lactose or isopropyl-beta-D-thiogalactoside (IPTG). So when the inhibition is relieved, the sgRNA will transcribe successfully and combine with Cas9 to cut the target gene panD. That means the E. coli won’t survive. We can demonstrate the difference of the transcriptional level of sgRNA by analyzing the growth curve in four different conditions (① +Ara +IPTG ② +Ara -IPTG ③ -Ara +IPTG ④ -Ara -IPTG)

Lysis

The aim of our project is cutting ARGs in engineered bacteria and reduce ARGs. To achieve this goal, we used the part BBa_K2277000, which had been constructed by 2017 iGEM team ZJUT-China. But in most cases, ARGs have been added to plasmids in order to screen transformants. So we integrated the lysin gene directly into the genome. Firstly, it can reduce the number of plasmids that need to be transformed in the engineering bacteria, which can reduce the additional metabolic pressure. Secondly, it can avoid the addition of new resistance.

The arabinose-induced promoter offers the possibility to regulate the expression of lysin protein in E. coli by adding or not adding a certain amount of arabinose into the culture, therefore control the lysin system.

Experiment design:

We first built this part on plasmid T vector. To verify whether the expressed lysin protein has the function of cell lysis, we used E. coli MG1655 as a chassis. we transformed this plasmid into MG1655 and characterized the function of the lysin gene based on the growth curve of the bacteria.

When the function of lysin protein was verified, we started to construct this part in the genome of E.coil MG1655 by using CRISPR/Cas9 system. In this system, pTargetF plasmid will express sgRNA which guide Cas9 protein cut target gene, causing double-strand DNA break. At this point, the donor DNA with the homologous arms will be inserted into the cutting site by homologous recombination. We designed the pTargetF plasmid which can transcribe the sgRNA targeting at specified site in the genome. We also constructed the donor DNA, which is composed of AraC-P_BAD-lysis and two homologous arms of the genome of MG1655. After construction, we characterized the function of the lysin gene based on cell growth on plates and in liquid culture.

The experiment is divide into 4 steps:

1.Construct T-MG1655 plasmid (contain 2000bp homologous arm of genome) by TA Cloning

2. Construct T-MG1655-Lysis plasmid by one-step cloning

3. Construct pTargetF-Lysis plasmid targeting E. coli MG1655 genome

4. Transform pCas and pTargetF-Lysis and fragment of AraC-P_BAD-Lysin into E. coliMG1655 to construct th desired recombinant strain

You can gain more details from our notebook

@@ Line 686: / Line 686: @@
 }
 p{
    text-indent:2em!important;
 }
@@ Line 1,027: / Line 1,027: @@
      </h1>
      <p>
-         The antimicrobial resistance is mostly caused by the expression of related antibiotic resistance genes(ARGs), which are widely being used as screening markers. But no effective method has been applied to ensure that resistance genes would not be released into the environment yet. This situation poses a great threat to the society and is exactly the problem we want to solve.
+         The antimicrobial resistance is mostly caused by the expression of related antibiotic resistance genes(ARGs), which are widely being used as screening markers. But no effective method has been applied to ensure that ARGs would not be released into the environment yet. This situation poses a great threat to the society and is exactly the problem we want to solve.
-     <br>The goal of our project is to eliminate the ARGs carried by the plasmids of the bacteria themselves. The way of elimination is to cleave the target resistance gene by the light-controlled expression of the Cas9 protein through the guidance of sgRNA. So we designed the following gene circuits:
+     <br>The goal of our project is to eliminate the ARGs carried by the plasmids of the bacteria themselves. The way of elimination is to cleave the target ARGs by the light-controlled expression of the Cas9 protein through the guidance of sgRNA. So we designed the following gene circuits:
      </p>
      <div class="designimg">
@@ Line 1,034: / Line 1,034: @@
      </div>
      <p>
-       Our goal is to eliminate the ARGs carried on the plasmids used in laboratories and fermentation plants, but it is not realistic to build our system on this plasmid because our system is too huge. So weould need a second plasmid as a vector for the light-controlled cleavage system. In this case we plan to move the different ARGs on the different two plasmids, and achieve their occurrence in a certain order. To this end, we have improved the original design:
+       Our goal is to eliminate the ARGs carried on the plasmids used in laboratories and fermentation plants. But it is difficult to build our system on this plasmid because our system is too huge. So weould need a second plasmid as a vector for the light-controlled cleavage system. In this case we plan to move the different ARGs on the different two plasmids, and achieve their occurrence in a certain order. To this end, we have improved the original design:
      </p>
      <div class="designimg">
@@ Line 1,040: / Line 1,040: @@
      </div>
      <p>
-       SgRNA1 transcribed by guide1 will guide Cas9 to cleave ARG1 on Plasmid 1, and sgRNA 2 transcribed by guide2 will guide Cas9 to cleave ARG2 on Plasmid 2. When ARG1 on Plasmid 1 is not cleaved, the plasmid is intact, and the normally expressed Repressor will repress the transcription of guide2; when ARG1 is uncleaved, Repressor would not be expressed, resulting in the transcription of guide2. This leads to the Cas9 cutting ARG2 on Plasmid 2. Our design allows the two plasmids to be cleaved in a certain order so that self-destruction would follow the elimination of the target gene.
+       The sgRNA1 transcribed by the guide1 will guide Cas9 to cleave ARG1 on the plasmid1, and the sgRNA2 transcribed by the guide2 will guide Cas9 to cleave ARG2 on Plasmid 2. When ARG1 on Plasmid 1 is not cleaved, the plasmid is intact, and the normally expressed Repressor will repress the transcription of the guide2. when ARG1 is cleaved, Repressor cannot be expressed normally, resulting in the transcription of the guide2. This leads to ARG2 degradation by Cas9 on the plasmid2. Our design allows the two plasmids to be cleaved with a certain order to ensure self-destruction after elimination of the target gene.
      </p>
      <div class="designimg">
@@ Line 1,154: / Line 1,154: @@
            <h2>1.1.Introduction of primary light control system</h2>
            <p>
-             The primary light control system is shown in figure 1. Under dark condition, the phosphate group is transferred from Yf1 protein to FixJ protein, the phosphorylated FixJ protein activates the P<sub>fixK2</sub> promoter and then activates the downstream gene expression. When induced by light, the phosphorylation of Fixj protein will be blocked, and the expression of genes regulated by P<sub>fixK2</sub> promoter will be inhibited.
+             The primary light control system is shown in figure 1. Under dark condition, the phosphate group is transferred from Yf1 protein to FixJ protein, the phosphorylated FixJ protein activates the P<sub><i>fixK2</i></sub> promoter and then activates the downstream gene expression. When induced by light, the phosphorylation of Fixj protein will be blocked, and the expression of genes regulated by P<sub><i>fixK2</i></sub> promoter will be inhibited.
            </p>
            <div class="designimg">
@@ Line 1,162: / Line 1,162: @@
            <h2>1.2.Experimental design</h2>
            <p>
-             To further verify the effectiveness of the system, we plan to transfer plasmids (dusk-eGFP-pUC57) with the system to different hosts. Cultured under blue and dark conditions after a period of time, then detected the fluorescence value of eGFP. The efficiency of primary light control system in different hosts was compared. The flow of the experiment is shown in figure 2.
+             To further verify the effectiveness of the system, we plan to transfer plasmids (dusk-eGFP-pUC57) with the system to different hosts. Cultured under blue and dark conditions after a period of time, then detected the fluorescence value of eGFP. The efficiency of primary light control system in different hosts was compared.
            </p>
-          <div class="designimg">
-            <img src="https://static.igem.org/mediawiki/2018/d/d6/T--ZJUT-China--designyu2.png" alt="">
-          </div>
-          <div class="note">
-            Figure 2. experimental flow
-          </div>
            <h2>1.3.Experimental method</h2>
            <h2>1.3.1.Extraction of dusk-eGFP-pUC57 plasmid</h2>
-           <p>Because the 2017 ZJUT team had already constructed the dusk-eGFP-pUC57 plasmid, we got the strain containing the plasmid from them and transferred it to a shaking flask containing 50ml LB liquid medium for overnight culture, and then extracted the plasmids using the DNA extraction kit. See the specification of the kit for specific operation.
+           <p>Because the 2017 ZJUT team had already constructed the dusk-eGFP-pUC57 plasmid,we used a shaking flask containing 50ml LB liquid medium to incubate bacteria carrying the plsmid overnight, and then extracted the plasmids using the DNA extraction kit. See the specification of the kit for specific operation.
            </p>
            <h2>1.3.2.Preparation of competent cells</h2>
@@ Line 1,204: / Line 1,198: @@
            <h2>2.2.Experimental design</h2>
            <p>
-             We plan to obtain the T7 promoter from the pET28b plasmid, delete the PJ23100 promoter from the dusk-eGFP-pUC57 plasmid, construct the T7-dusk-eGFP-pUC57 plasmid by one step clone method, transfer it into the BL21 host and induce it by adding different amount of IPTG. The expression of eGFP was detected by culturing under blue light and dark light for a period of time. The flow of the experiment is shown in figure 4.
+             We plan to obtain the T7 promoter from the pET28b plasmid, delete the PJ23100 promoter from the dusk-eGFP-pUC57 plasmid, construct the T7-dusk-eGFP-pUC57 plasmid by one step clone method, transfer it into the BL21 host and induce it by adding different amount of IPTG. The expression of eGFP was detected by culturing under blue light and dark light for a period of time.
            </p>
-          <div class="designimg">
-            <img src="https://static.igem.org/mediawiki/2018/1/16/T--ZJUT-China--designyu4.png" alt="">
-          </div>
-          <div class="note">Figure 4. experimental flow</div>
            <h2>2.3.Experimental method</h2>
            <h2>2.3.1.Acquisition of T7 Promoter</h2>
@@ Line 1,346: / Line 1,336: @@
            </p>
            <h2>
-.3.3.De-template
+.3.3.Template elimination
            </h2>
            <p>
-             PCR products need to be treated with Dpn Ⅰ enzyme to remove the effect of template plasmids on subsequent experiments. The dosage is as follows:
+             PCR products need to be treated with Dpn Ⅰ enzyme to remove template plasmids on subsequent experiments. The reaction system is as follows:
            </p>
            <div class="designimg">
@@ Line 1,355: / Line 1,345: @@
            </div>
            <p>
-             De-template results were detected by electrophoresis gel.
+             Result of template elimination were detected by electrophoresis gel.
            </p>
            <h2>
@@ Line 1,361: / Line 1,351: @@
            </h2>
            <p>
-             The insertion fragments and linearized carriers were purified by purification kit, then connected by one-step cloning kit, and then transferred into BL21 competent cells for overnight culture at 37 ℃. Observe the number of transformants. The dosage of one-step cloning is as follows:
+             The insertion fragments and linearized carriers were purified by purification kit, then connected by one-step cloning kit, and then transferred into BL21 competent cells for overnight culture at 37 ℃. The number of transformants were counted. The reaction system of one-step cloning is as follows:
            </p>
            <div class="designimg">
@@ Line 1,367: / Line 1,357: @@
            </div>
            <h2>2.3.4.Secondary light control system effect detection</h2>
-           <p>The successful transformants were added to different amounts of IPTG, and cultured under blue light and dark for 24 hours to detect the expression of eGFP.
+           <p>The successful transformants were added to different amounts of IPTG, and cultured under blue light and dark conditions for 24 hours to detect the expression of eGFP.
            </p>
            <button type="button" name="button"id="btn2">Back</button>
@@ Line 1,374: / Line 1,364: @@
          <div id="div21">
-           <h1>Constructing a plasmid expressing sgRNA which target chloramphenicol <br><br>resistance gene</h1>
+           <h1>Constructing a plasmid expressing sgRNA which target chloramphenicol <br><br>resisitance gene</h1>
-           <h2>▶First step: Test Crisper/Cas9 system</h2>
+           <h2>▶First step: Test CRISPR/Cas9 system</h2>
            <h2>Overview</h2>
            <p>
-             In order to achieve degradation of antibiotic resistance genes, we have been trying to construct a resistant cutting system by using Crisper/Cas9 system. In this system, plasmid ptargetF expresses sgRNA, to guide cas9 protein to cleave target gene. Firstly, We have been testing Crisper/Cas9 system. We selected two different kinds of <i>E.coli</i>. One is wild type, the other is panD mutant (there is no panD gene in genome). We made the two strains into receptive state and introduced pCas plasmid and pTargetF plasmid respectively. Finally, the wild type died and the panD mutant grew. It is proved that the CRISPER/Cas9 system is effective.
+             We constructed a CRISPR/Cas9 system for degrading ARGs. In this system, plasmid pTargetF expresses sgRNA, to guide Cas9 protein to cleave target gene. Firstly, We tested the CRISPR/Cas9 system. We selected two different kinds of <i>E. coli</i>.The one is wild type, the other is a <i>panD</i> mutant. We transferred pCas plasmid and pTargetF plasmid into the two strains. We observed that the wild type died and the <i>panD</i> mutant grew. The results proved that the CRISPR/Cas9 system was effective.
            </p>
            <h2>
@@ Line 1,384: / Line 1,374: @@
            </h2>
            <p>
-             The plasmid pTargetF-panD expresses sgRNA which targets on the panD . In a panD mutant E.coli MG1655, the bacteria constructed in our lab. Therefore, pTargetF-panD can guide Cas9 to the panD gene and cause double-strand DNA break in the genome of <i>E.coli</i> MG1655.
+             The plasmid pTargetF-<i>panD</i> expresses sgRNA which targets on the <i>panD</i>, which guides Cas9 to the <i>panD</i> gene and cause double-strand DNA break in the genome of <i>E. coli</i> MG1655.
            </p>
            <h2>Functional verification</h2>
            <p>
-             The strain we used is <i>E.coli</i> MG1655+ΔpanD and <i>E.coli</i> MG1655 wild type. Firstly, we need to prepare <i>E.coli</i> MG1655+ΔpanD and <i>E.coli</i> MG1655 wild type competent cells and transform pCas plasmid into it. Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-panD plasmid, respectively. Finally, the transformed plates were placed in 30℃ devices overnight.
+             The strain we used is <i>E. coli</i> MG1655 Δ<i>panD</i> and <i>E. coli</i> MG1655 wild type. Firstly, we need to prepare <i>E. coli</i> MG1655 Δ<i>panD</i> and <i>E. coli</i> MG1655 wild type competent cells and transform pCas plasmid into it. Then, we need to transform pTargetF-<i>panD</i> plasmid into the above transformants. Finally, the plates were incubated at 30℃ overnight.
            </p>
-           <p> pTargetF-panD can guide Cas9 to the panD gene and cause double-strand DNA break in the genome of <i>E.coli</i> MG1655 wide type, while pTargetF-panD can not guide Cas9 to any place in the genome of <i>E.coli</i> MG1655+ΔpanD. After the transformed plates placed in 30℃ devices overnight, <i>E.coli</i> MG1655+ΔpanD grew, <i>E.coli</i> MG1655 wild type died.
+           <p> pTargetF-<i>panD</i> can guide Cas9 to the <i>panD</i> gene and cause double-strand DNA break in the genome of <i>E. coli</i> MG1655 wide type, while pTargetF-<i>panD</i> can not guide Cas9 to any place in the genome of <i>E. coli</i> MG1655 Δ<i>panD</i>. Transformants of <i>E. coli</i> MG1655 Δ<i>panD</i> grew, Transformants of <i>E. coli</i> MG1655 wild type died.
            </p>
            <h2>▶Second step: Construct pTargetF-cm</h2>
            <h2>Overview</h2>
            <p>
-             In order to achieve degradation of antibiotic resistance genes, we have been trying to construct a resistant cutting system by using Crisper/Cas9 system. In this system, plasmid ptargetF expresses sgRNA, to guide cas9 protein to cleavage target gene. Secondly, We have been construct ptargetF-cm(plasmid can express sgRNA which targets chloramphenicol). And sequencing results showed construction successfully.
+             In order to achieve degradation of ARGs, we have been trying to construct a resistant cutting system by using CRISPR/Cas9 system. In this system, plasmid pTargetF expresses sgRNA, to guide Cas9 protein to the target gene. Secondly, We have been construct pTargetF-cm, which can express sgRNA which targets chloramphenicol resistance gene. And sequencing results showed successful construction.
            </p>
            <h2>
              Functional description
            </h2>
-           <p>The plasmid pTargetF-cm can expresses sgRNA which targets on the cm. </p>
+           <p>The plasmid pTargetF-cm can expresses sgRNA which targets on the chloramphenicol resistance gene. </p>
            <h2>Functional verification</h2>
-           <p> After PCR, we get the plasmid pTargetF-cm, then transform it into <i>E.coli</i> DH5α competent cells. Then, we need to culture the transformants and send it to sequence.
+           <p> After PCR, we got the plasmid pTargetF-cm, then transformed it into <i>E. coli</i> DH5α competent cells.The construction was confirmed by sequancing.
            </p>
            <h2>▶Third step: Test pTargetF-cm</h2>
            <h2>Overview</h2>
-           <p>In order to achieve degradation of antibiotic resistance genes, we have been trying to construct a resistant cutting system by using Crisper/Cas9 system. In this system, plasmid ptargetF expresses sgRNA, to guide cas9 protein to cleavage target gene. Thirdly, We have been testing the function of the plasmid pTargetF-cm. We choose <i>E.coli</i>.MG1655 wild type and made the strain into receptive state and introduced pSu(there is chloramphenicol resistance gene) plasmid, pCas plasmid(there is kanamycin resistance gene) and pTargetF plasmid(there is spectinomycin resistance gene) respectively. Culture the transformants. Finally, a transformant can grow in LB+kan+spec, while it died in LB+cm. The result showed pTargetF-cm working successfully.
+           <p>In order to achieve degradation of ARGs, we have been trying to construct a resistant cutting system by using CRISPR/Cas9 system. In this system, plasmid pTargetF expresses sgRNA, for guiding Cas9 protein to cleavage target gene. Thirdly, we tested the function of the plasmid pTargetF-cm.We introduced pSU20 which has a chloramphenicol resistance gene and pCas which has a kanamycin resistance gene into <i>E. coli</i> MG1655.
+          Finally, the transformant can grow in LB supplemented with kanamycin and spectinomycin, while it can not grow in LB supplemented with chloramphenicol. The result showed that pTargetF-cm worked successfully.
            </p>
            <h2>Functional description</h2>
            <p>
-             The plasmid pTargetF-cm expresses sgRNA which targets on the cm in <i>E.coli</i> MG1655-pSu(cm). Therefore, pTargetF-cm can guide Cas9 to the cm resistance gene and cause double-strand DNA break in the pSu plasmid.
+             The plasmid pTargetF-cm expresses sgRNA which targets on the chloramphenicol in <i>E. coli</i> MG1655-pSU20. Therefore, pTargetF-cm can guide Cas9 to the chloramphenicol resistance gene and cause double-strand DNA break in the pSU20 plasmid.
            </p>
            <h2>Functional verification</h2>
-           <p>The strain we used is <i>E.coli</i> MG1655 wild type. First, we need to prepare <i>E.coli</i> MG1655 wild type competent cells and transform pSu plasmid into it. Then, we need to culture the transformants and prepare them into competent cells, and then transform pCas plasmid. Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-cm plasmid, respectively. Finally, the transformed plates were placed in 30℃ devices overnight.
+           <p>The strain we used is <i>E. coli</i> MG1655 wild type. First, we need to prepare <i>E. coli</i> MG1655 wild type competent cells and transform pSU20 plasmid into it. Then, we need to culture the transformants and prepare them into competent cells, and then transform pCas plasmid. Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-cm plasmid, respectively.
            </p>
+            <p>pTargetF-cm can guide Cas9 to the chloramphenicol resistance gene and cause double-strand DNA break in the pSU20 plasmid. After grown at 30℃ overnight,After incubation at 30 C overnight, <i>E. coli</i> MG1655 wild type was able to grow in LB supplemented with kanamycin and spectinomycin but failed to grow in the culture supplemented with chloramphenicol because the corresponding resistance gene in pSU20 had been eliminated.
-            <p>pTargetF-cm can guide Cas9 to the cm resistance gene and cause double-strand DNA break in the pSu plasmid. After the transformed plates placed in 30℃ devices overnight, <i>E.coli</i>MG1655 wild type still has pCas plasmid and pTargetF-cm plasmid. So it can grow in  LB+kan+spec. However the pSu plasmid can’t work. So it died in LB+cm.
            </p>
            <button type="button" name="button"id="btn22">Back</button>
@@ Line 1,425: / Line 1,415: @@
          <div id="div31">
              <h1>P<sub>BAD</sub>-Cas9</h1>
-             <p>Before we used the light-controlled system to regulate the expression of cas9, we first used P<sub>BAD</sub> to regulate the expression of cas9 to verify the cleavage function of cas9.
+             <p>Before we used the light-controlled system to regulate the expression of Cas9, we first used P<sub>BAD</sub> to regulate the expression of Cas9 to verify the cleavage function of Cas9.
              </p>
              <div class="designimg">
@@ Line 1,431: / Line 1,421: @@
              </div>
              <p>
-               The arabinose-induced promoter offers the possibility to regulate the expression of Cas9 protein in <i>Escherichia coli</i> by adding or not adding a certain amount of arabinose into the culture, therefore control the CRISPR/Cas system.
+               The arabinose-induced promoter offers the possibility to regulate the expression of Cas9 protein in <i>E. coli</i> by adding or not adding a certain amount of arabinose into the culture.
              </p>
              <h2>Experiment design:</h2>
              <p>
-               We built this part on pGLO and named it pGLO-Cas9. To verify whether the expressed Cas9 protein has the function of cutting the target gene, we used <i>E.coli</i>MG1655 as a chassis. In addition, we will use the pTF plasmid to transcribe the sgRNA that targets the panD gene. Since the original pTF is not compatible with pGLO, we have built a pTF-p15A that is compatible with pGLO.
+               We built this part on pGLO and named it pGLO-Cas9. To verify whether the expressed Cas9 protein has the function of cutting the target gene, we used <i>E. coli</i> MG1655 as a chassis. In addition, we will use the pTargetF plasmid to transcribe the sgRNA that targets the <i>panD</i> gene. Since the original pTargetF is not compatible with pGLO, we have built a pTargetF-p15A that is compatible with pGLO.
                <a href="See our notebook for more details."></a> Finally, we characterized the function of the Cas9 gene based on the growth curve of the bacteria.
-               <br>When the function of Cas9 was verified, we replaced the sgRNA targeting panD with the sgRNA targeting the chloramphenicol gene. The plasmid we constructed was named pTF-p15A-cm. And we used <i>E.coli</i> MG1655 containing the chloramphenicol gene on genome for validation experiments.
+               <br>When the function of Cas9 was verified, we replaced the sgRNA targeting <i>panD</i> with the sgRNA targeting the chloramphenicol gene. The plasmid we constructed was named pTargetF-p15A-cm. And we used <i>E. coli</i> MG1655 containing the chloramphenicol gene on genome for validation experiments.
                <h2>The experiment is divide into 5 steps:</h2>
                <p>
-                 the regulation of an inducible promoter. This is based on a given plasmid pTF, we changed its replicon to make it compatible with P<sub>BAD</sub>-Cas9.
+                 the regulation of an inducible promoter. This is based on a given plasmid pTargetF, we changed its replicon to make it compatible with P<sub>BAD</sub>-Cas9.
-              <br> 3) Transform both plasmid to <i>E. coli</i> MG1655
+                <br>  1) Construct the P<sub>BAD</sub>-Cas9 plasmid based on a given plasmid pGLO.
-              <br>4) Get the growth curve result to prove after adding arabinose, the Cas9 protein will express and perform its function.
+                <br>  2) Construct a plasmid pTargetF-p15A to express sgRNA to test if the Cas9 protein is function under the regulation of an inducible promoter. This is based on a given plasmid pTargetF, we changed its replicon to make it compatible with P<sub>BAD</sub>-Cas9.
-                <br>5) Construct and test a plasmid pTF-p15A-cm to perform resistance gene cut.
+                <br> 3) Transform both plasmid to <i>E. coli</i> MG1655
+                <br>4) Get the growth curve result to prove after adding arabinose, the Cas9 protein will express and perform its function.
+                <br>5) Construct and test a plasmid pTargetF-p15A-cm to perform ARGs cut.
                 <br>
-                 *panD gene is a gene on the genome of <i>E. coli</i>, in the previous research of our lab, we obtained a panD-deficient strain of <i>E. coli.</i>
+                 *<i>panD</i> gene is a gene on the genome of <i>E. coli</i>, in the previous research of our lab, we obtained a <i>panD</i>-deficient strain of <i>E. coli.</i>
                </p>
              </p>
@@ Line 1,456: / Line 1,448: @@
            <h2> 1.1Functional description</h2>
            <p>
-             YF1 is a fusion protein of a LOV protein domain and histidine kinase. In the dark YF1 phosphorylates FixJ response regulator and phospholylated FixJ response regulator activates P<sub>fixk2</sub> promoter and then activates the Cas9 gene expression. In blue light YF1 don’t phosphorylate FixJ response regulator, so P<sub>fixk2</sub> promoter isn’t activated,and the expression of gene regulated by P<sub>fixK2</sub> promoter will be inhibited.
+             Under the dark condition, YF1 phosphorylates FixJ and phospholylated FixJ activates the P<sub><i>fixK2</i></sub> promoter, which activates the expression of Cas9.Under the blue light condition YF1 don’t phosphorylate FixJ. Therefore P<sub><i>fixK2</i></sub> promoter can not initiate the expression of Cas9.
            </p>
+           <p>The plasmid pTargetF-cm expresses sgRNA which targets on the chloramphenicol resistance gene . In a <i>panD</i> mutant <i>E. coli</i> Bl21+Δ<i>panD</i>, the original <i>panD</i> gene was replaced by the chloramphenicol resistance gene in the genome. Therefore, pTargetF-cm can guide Cas9 to the chloramphenicol gene and cause double-strand DNA break in the genome of <i>E. coli</i> Bl21+Δ<i>panD</i>.
+           </p><p>Under dark conditions, <i>cas9</i> gene can be expressed, so pTargetF-cm can guide Cas9 to the chloramphenicol resistance gene and cause double-strand DNA break in the genome.However, in blue light,the expression of Cas9 is inhibited, double-strand DNA will not be cut.
-           <p>The plasmid pTargetF-cm expresses sgRNA which targets on the chloramphenicol resistance gene (cm). In a panD mutant <i>E.coli</i> Bl21+ΔpanD, the original panD gene was replaced by the cm gene in the genome. Therefore, pTargetF-cm can guide Cas9 to the cm gene and cause double-strand DNA break in the genome of <i>E.coli</i> Bl21+ΔpanD.
-           </p><p>Under dark conditions, Cas9 gene can be expressed,so pTargetF-cm can guide Cas9 to the cm gene and cause double-strand DNA break in the genome.However,in blue light,the expression of Cas9 gene is inhibited,double-strand DNA will not be cut.
            </p>
            <div class="designimg">
              <img src="https://static.igem.org/mediawiki/2018/4/47/T--ZJUT-China--designmao1.png" alt="">
            </div>
-           <div class="note">Figure 1. Light-controlled system+Cas9</div>
+           <div class="note">Figure 1. Light-controlled CRISPR/Cas9 system</div>
            <h2>1.2Experimental design</h2>
            <h2>1.2.1Design of plasmid needed </h2>
            <p>
-             In this experiment,we need dusk-Cas9-pUC57 plasmid,pTargetF-cm plasmid,pSU20 plasmid and pTargetF-panD plasmid.We borrowed dusk-eGFP-pUC57 plasmid,pSU20 plasmid and pTargetF-panD plasmid from our sisters,and we used dusk-eGFP-pUC57 plasmid and pTargetF-panD plasmid to construct dusk-Cas9-pUC57 plasmid and pTargetF-cm plasmid,using PCR technology.For detailed experimental procedure,
+             In this experiment, we need dusk-Cas9-pUC57 plasmid,pTargetF-cm plasmid, pSU20 plasmid and pTargetF-<i>panD</i> plasmid. We used dusk-eGFP-pUC57 plasmid and pTargetF-<i>panD</i> plasmid to construct dusk-Cas9-pUC57 plasmid and pTargetF-cm plasmid. For detailed experimental procedure,
               please refer to our notebook.
                <a href="https://static.igem.org/mediawiki/2018/c/c6/T--ZJUT-China--mao714.pdf">7.14</a>
@@ Line 1,480: / Line 1,470: @@
            </h2>
            <p>
-             The strain we used is <i>E.coli</i> Bl21+ΔpanD,which is a panD mutant, the original panD gene was replaced by the chloramphenicol resistance gene in the genome(cm).First, we need to prepare <i>E.coli</i> Bl21+ΔpanD competent cells and transform dusk-Cas9-pUC57 plasmid into it.Then, we need to culture the transformants and prepare them into competent cells, and then transform pTargetF-cm plasmid,pSU20 plasmid (as control) and pTargetF-panD plasmid (as control) into it,respectively.Finally, the transformed plates were placed in blue and dark devices overnight.
+             The strain we used is <i>E. coli</i> BL21 Δ<i>panD</i>, which is a <i>panD</i> mutant, the original <i>panD</i> gene was replaced by the chloramphenicol resistance gene in the genome(cm). First, we need to prepare <i>E. coli</i> BL21 Δ<i>panD</i> competent cells and transform dusk-Cas9-pUC57 plasmid into it.Then, we transformed pTargetF-cm plasmid, pSU20 plasmid (as control) and pTargetF-<i>panD</i> plasmid (as control) into <i>E. coli</i> BL21 Δ<i>panD</i> dusk-Cas9-pUC57 separately. The transformants were incubated overnight under dark or blue light conditions.
-           </p><p>  Under dark conditions,pTargetF-cm can guide Cas9 to the cm gene and cause double-strand DNA break in the genome of <i>E.coli</i> Bl21+ΔpanD, while pTargetF-panD can not guide Cas9 to any place in the genome of E.coli Bl21+ΔpanD. The damage to the bacterial genome due to CRISPR/Cas9 can be determined by transformation efficiency. When light was provided, the light-controlled gene expression system was suppressed and therefore the transformation efficiency was higher.
+           </p><p>  Under dark conditions, pTargetF-cm can guide Cas9 to the chloramphenicol gene and cause double-strand DNA break in the genome of <i>E. coli</i> Bl21 Δ<i>panD</i>, while pTargetF-<i>panD</i> can not guide Cas9 to any place in the genome of <i>E. coli</i> Bl21 Δ<i>panD</i>. The damage to the bacterial genome due to CRISPR/Cas9 can be determined by transformation efficiency. When light was provided, the light-controlled gene expression system was suppressed and therefore the transformation efficiency was higher.
            </p>
              <div class="designimg">
@@ Line 1,496: / Line 1,486: @@
            <h2>Overview</h2>
            <p>
-             In order to achieve the sequential degradation of antibiotic resistance genes (ARGs), we have been trying to construct a repression system. In this system, the arabinose-controlled repressor protein was utilized to regulate the transcription of sgRNA. We chose two types of inhibitor-promoter: lacI-P<sub>lac</sub>, cI-P<sub>R</sub> to test just in case. The LacI repressor is a DNA-binding protein which can bind to the lac operator to inhibit transcription in <i>E.coli</i>. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG). The P<sub>R</sub> promoter has two DNA binding sites for cI repressor. And transcription would be repressed when cI protein binds to the sites. After experiment, we selected the lacI-Plac to make up the repression system due to the results.
+             In order to achieve the sequential degradation of ARGs, we have been trying to construct a repression system. In this system, the arabinose-controlled repressor protein was utilized to regulate the transcription of sgRNA. We chose two types of inhibitor-promoter: <i>lacI</i>-P<sub><i>lac</i></sub>, <i>cI</i>-P<sub>R</sub> to test just in case. The LacI repressor is a DNA-binding protein which can bind to the <i>lac</i> operator to inhibit transcription in <i>E. coli</i>. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG). The P<sub>R</sub> promoter has two DNA binding sites for CI repressor. And transcription would be repressed when CI protein binds to the sites. After experiment, we selected the <i>lacI</i>-P<sub><i>lac</i></sub> to make up the repression system due to the results.
            </p>
            <h2>Design</h2>
-           <h2>(1)lacI-P<sub>lac</sub></h2>
+           <h2>(1)<i>lacI</i>-P<sub><i>lac</i></sub></h2>
            <p>The genetic circuit is shown in Fig.1.</p>
            <div class="designimg">
              <img src="https://static.igem.org/mediawiki/2018/5/5f/T--ZJUT-China--design51.png" alt="">
            </div>
-             <div class="note"> Fig.1 The genetic circuit of lacI-Plac</div>
+             <div class="note"> Fig.1 The genetic circuit of lacI-P<sub><i>lac</i></div></sub>
              <p>
                This part is mainly composed of three elements:
-             </p><p>①Arabinose induced promoter P<sub>BAD</sub> to express the gene of downstream. This part contains the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction. (“upstream”) By binding to L (+)-arabinose, AraC changes its conformation. This causes the protein to diffuse from the DNA thereby inducing transcription.
+             </p><p>①Arabinose induced promoter P<sub>BAD</sub> to express the gene of downstream. This part contains the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction.By binding to L (+)-arabinose, AraC changes its conformation and induce the transcription of downstream genes.
-             </p><p>②This part contains the lac operator as well as the coding sequence for the repressor lacI. The lacI repressor binds to the lac operator to inhibit transcription in <i>E.coli</i>. This inhibition can be relieved by adding lactose or isopropyl -beta -D-thiogalactopyranoside (IPTG).
+             </p><p>②This part contains the <i>lac</i> operator as well as the coding sequence for the repressor lacI. The lacI repressor binds to the <i>lac</i> operator to inhibit transcription in <i>E. coli</i>. This inhibition can be relieved by adding lactose or isopropyl -beta -D-thiogalactopyranoside (IPTG).
-              </p><p>③GFP, as the report gene, can verify the degree of repression by repressor.
+            </p><p>③GFP, as the report gene, can show the degree of repression by repressor.
              </p>
            <h2>（2）cI-P<sub>R</sub></h2>
@@ Line 1,523: / Line 1,513: @@
            </p>
            <h2>Description</h2>
-           <p>Different concentrations of arabinose will induce these parts with different concentration of repressor proteins. By adding different concentration of arabinose, we can measure the fluorescence intensity of GFP and find out the relationship between the concentration of arabinose and RFU (Relative fluorescence unit) intensity. And by adding IPTG，the LacI protein is able to be released from the lac operator .It is a characteristic that can be utilized to further verify the function of the lacI-P<sub>lac</sub> by adding IPTG or not.
+           <p>Different concentrations of arabinose will induce these parts with different concentration of repressor proteins. By adding different concentration of arabinose, we can measure the fluorescence intensity of GFP and find out the relationship between the concentration of arabinose and RFU (Relative fluorescence unit) intensity. And by adding IPTG，the LacI protein is able to be released from the <i>lac</i> operator .It is a characteristic that can be utilized to further verify the function of the lacI-P<sub><i>lac</i></sub> by adding IPTG or not.
            </p>
            <h2>▶Second step</h2>
            <h2>Overview</h2>
            <p>
-             Based on the results we have achieved, we chose lacI-P<sub>lac</sub> to carry out the next step. In this system, the arabinose-controlled repressor protein is utilized to regulate the transcription of sgRNA.
+             Based on the results we have achieved, we chose <i>lacI</i>-P<sub><i>lac</i></sub> to carry out the next step. In this system, the arabinose-controlled repressor protein is utilized to regulate the transcription of sgRNA.
-           </p><p> Single-guide RNA (sgRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It contains the 20-bp complementary region (N20) with the requisite NGG PAM matching genomic loci of interest and the sequence of tracrRNA. It will guide the nuclease Cas protein 9(Cas9) to the target. It is of great importance in CRISPR/Cas9 system. We design the sgRNA which specifically targets the gene panD on the <i>E. coli</i> genome in order to demonstrate our CRISPR/Cas9 system.
+           </p><p> Single-guide RNA (sgRNA) is an artificial RNA which is designed to bind a certain DNA sequence. It contains the 20-bp complementary region (N20) with the requisite NGG PAM matching genomic loci of interest and the sequence of tracrRNA. It will guide Cas9 to the target. We design the sgRNA which specifically targets the gene <i>panD</i> on the <i>E. coli</i> genome in order to demonstrate our CRISPR/Cas9 system.
            </p>
-           <h2>●lacI-P<sub>lac</sub>-sgRNA</h2>
+           <h2>●lacI-P<sub><i>lac</i></sub>-sgRNA</h2>
            <p>The genetic circuit is shown in Fig.3.</p>
            <div class="designimg">
              <img src="https://static.igem.org/mediawiki/2018/2/2e/T--ZJUT-China--design53.png" alt="">
            </div>
-           <div class="note"> Fig.3 The genetic circuit of lacI-Plac-sgRNA</div>
+           <div class="note"> Fig.3 The genetic circuit of <i>lacI</i>-P<i>lac</i>-sgRNA</div>
            <p>
              We combined lacI repression system (BBa_K2556031) which is controlled by the AraC-Ara promoter (BBa_K808000) with sgRNA (BBa_K2556041) so that we can regular the transcription of sgRNA. It’s important to activate the CRISPR/Cas9 system only under certain proper conditions.
@@ Line 1,542: / Line 1,532: @@
            <h2>Description</h2>
            <p>
-             When activating the AraC-Ara promoter by adding L (+)-arabinose, the LacI repressor will express and the transcription of sgRNA will be inhibited. But this inhibition can be relieved after induction with lactose or isopropyl-beta-D-thiogalactoside (IPTG). So when the inhibition is relieved, the sgRNA will transcribe successfully and combine with Cas9 to cut the target gene panD. That means the <i>E. coli</i> won’t survive. We can demonstrate the difference of the transcriptional level of sgRNA by analyzing the growth curve in four different conditions (①+Ara +IPTG ②+ Ara ③+ IPTG ④None)
+             When activating the AraC-Ara promoter by adding L (+)-arabinose, the LacI repressor will express and the transcription of sgRNA will be inhibited. But this inhibition can be relieved after induction with lactose or isopropyl-beta-D-thiogalactoside (IPTG). So when the inhibition is relieved, the sgRNA will transcribe successfully and combine with Cas9 to cut the target gene <i>panD</i>. That means the <i>E. coli</i> won’t survive. We can demonstrate the difference of the transcriptional level of sgRNA by analyzing the growth curve in four different conditions (① +Ara +IPTG ② +Ara -IPTG ③ -Ara +IPTG  ④ -Ara -IPTG)
            </p>
              <button type="button" name="button"id="btn52">Back</button>
@@ Line 1,550: / Line 1,540: @@
          <div id="div61">
            <h1>Lysis</h1>
-             <p>The aim of our project is that cut antibiotic resistance gene in engineering bacteria and reduce resistance genes in industrial emissions. And in our final design, cells end up with a inducible-lysis after ARGs were cut. Then we find the part<a href="http://parts.igem.org/Part:BBa_K2277000">BBa_K2277000</a>, which constructed by 2017 iGEM team ZJUT-China. They construct the <i>lysin</i> gene in the plasmid. But in most cases, resistance genes will be added to plasmids in order to screen transformants. So we choose to construct the <i>lysin</i> gene directly into the genome,for two advantages. Firstly, it can reduce the number of plasmids that need to be transformed in the engineering bacteria, which can reduce the additional metabolic pressure. Secondly, it can avoid the addition of new resistance.
+             <p>The aim of our project is cutting ARGs in engineered bacteria and reduce ARGs. To achieve this goal, we used the part <a href="http://parts.igem.org/Part:BBa_K2277000">BBa_K2277000</a>, which had been constructed by 2017 iGEM team ZJUT-China. But in most cases, ARGs have been added to plasmids in order to screen transformants. So we integrated the <i>lysin</i> gene directly into the genome. Firstly, it can reduce the number of plasmids that need to be transformed in the engineering bacteria, which can reduce the additional metabolic pressure. Secondly, it can avoid the addition of new resistance.
              </p>
              <div class="designimg">
                <img src="https://static.igem.org/mediawiki/2018/3/3f/T--ZJUT-China--partl1.png" alt="">
              </div>
-             <p>The arabinose-induced promoter offers the possibility to regulate the expression of lysin protein in <i>E.coli</i> by adding or not adding a certain amount of arabinose into the culture, therefore control the lysin system.</p>
+             <p>The arabinose-induced promoter offers the possibility to regulate the expression of lysin protein in <i>E. coli</i> by adding or not adding a certain amount of arabinose into the culture, therefore control the lysin system.</p>
              <h2>Experiment design:</h2>
              <p>
-               We first built this part on plasmid T vector. To verify whether the expressed lysin protein has the function of cell lysis, we used <i>E.coli</i> MG1655 as a chassis. we transformed this plasmid into MG1655 and characterized the function of the <i>lysin</i> gene based on the growth curve of the bacteria.
+               We first built this part on plasmid T vector. To verify whether the expressed lysin protein has the function of cell lysis, we used <i>E. coli</i> MG1655 as a chassis. we transformed this plasmid into MG1655 and characterized the function of the <i>lysin</i> gene based on the growth curve of the bacteria.
-             </p><p>When the function of lysin protein was verified, we started to construct this part in the genome of <i>E.coil</i> MG1655 by using CRISPR/Cas9 system. In this system, pTargetF plasmid will express sgRNA which guide Cas9 protein cut target gene, causing double-strand DNA break. At this point, the donor DNA with the homologous arm will be inserted into the cut site according to homologous recombination. Then the gene was inserted successfully. We looked for an insertion point in the non-metabolic pathway of the <i>E.coli</i> MG1655 genome. Based on this site, we designed the pTargetF plasmid which can transcribe the sgRNA targeting that site in the genome. We also constructed the donor DNA, which is composed of AraC-P<sub>BAD</sub>-lysis and two genome homologous arm of the genome of MG1655. After constructed successfully, we characterized the function of the <i>lysin</i> gene based on plate culture and the growth curve of the bacteria.
+             </p><p>When the function of lysin protein was verified, we started to construct this part in the genome of <i>E.coil</i> MG1655 by using CRISPR/Cas9 system. In this system, pTargetF plasmid will express sgRNA which guide Cas9 protein cut target gene, causing double-strand DNA break. At this point, the donor DNA with the homologous arms will be inserted into the cutting site by homologous recombination. We designed the pTargetF plasmid which can transcribe the sgRNA targeting at specified site in the genome. We also constructed the donor DNA, which is composed of AraC-P<sub>BAD</sub>-lysis and two homologous arms of the genome of MG1655. After construction, we characterized the function of the <i>lysin</i> gene based on cell growth on plates and in liquid culture.
              </p>
              <h2>The experiment is divide into 4 steps:</h2>
-             <p>1.Construct T-MG1655 plasmid(contain 2000bp homologous arm of genome) by original TA Cloning</p>
+             <p>1.Construct T-MG1655 plasmid (contain 2000bp homologous arm of genome) by TA Cloning</p>
              <p>2. Construct T-MG1655-Lysis plasmid by one-step cloning</p>
-             <p>3. Construct of p-TargetF-Lysis plasmid targeting E.coli MG1655 Genome by PCR</p>
+             <p>3. Construct pTargetF-Lysis plasmid targeting <i>E. coli</i> MG1655 genome</p>
-             <p>4. Transform plasmids of pCas and p-TargetF-Lysis and fragment of AraC-P<sub>BAD</sub>-Lysis into <i>Eoli</i>.MG1655 to get target strain</p>
+             <p>4. Transform pCas and pTargetF-Lysis and fragment of AraC-P<sub>BAD</sub>-Lysin into <i>E. coli</i>MG1655 to construct th desired recombinant strain</p>
              <p>You can gain more details from our <a href="https://2018.igem.org/Team:ZJUT-China/Notebook">notebook</a></p>
              <button type="button" name="button"id="btn62">Back</button>
                  <p><br></p>

Difference between revisions of "Team:ZJUT-China/Design"

Revision as of 14:08, 17 October 2018

Team:ZJUT-China

Overview

Light-controlled system

1.Primary light control system

1.1.Introduction of primary light control system

1.2.Experimental design

1.3.Experimental method

1.3.1.Extraction of dusk-eGFP-pUC57 plasmid

1.3.2.Preparation of competent cells

1.3.3.Transformation

1.3.4.Primary light control system effect detection

2.Improvement of primary light control system

2.1.Replacement of a strong promoter

2.2.Experimental design

2.3.Experimental method

2.3.1.Acquisition of T7 Promoter

2.3.2.Delete J23100 promoter from dusk-eGFP-pUC57 plasmid

2.3.3.Template elimination

2.3.3.One-step cloning and transformation

2.3.4.Secondary light control system effect detection

Constructing a plasmid expressing sgRNA which target chloramphenicol resisitance gene

▶First step: Test CRISPR/Cas9 system

Overview

Functional description

Functional verification

▶Second step: Construct pTargetF-cm

Overview

Functional description

Functional verification

▶Third step: Test pTargetF-cm

Overview

Functional description

Functional verification

PBAD-Cas9

Experiment design:

The experiment is divide into 5 steps:

Light-controlled Cas9 system

1.1Functional description

1.2Experimental design

1.2.1Design of plasmid needed

1.2.2Functional verification

Repression system

▶First step

Overview

Design

(1)lacI-Plac

（2）cI-PR

Description

▶Second step

Overview

●lacI-Plac-sgRNA

Description

Lysis

Experiment design:

The experiment is divide into 4 steps:

Contact

Constructing a plasmid expressing sgRNA which target chloramphenicol

resisitance gene

P_BAD-Cas9

(1)lacI-P_lac

（2）cI-P_R

●lacI-P_lac-sgRNA