Investigation of the best gene order of phaCAB  for PHB production(Pepper)

Improving the production of PHBV by introducing bktB  to E. coli (Ming)

Investigation of the effect of phasin autoregulation system on PHB production (Qihui)

Construction of Plasmids Harbouring the Sleeping Beauty Mutase Operon and Methylmalonyl CoA Epimerase for Producing Propionate for PHBV Production (Craig)

Week 1 – Week 2

• Discuss the project aims and decide individual project strategy (shown in Figure 1).



Figure 1 Proposed mechanism for propionate synthesis utilising the Sleeping beauty mutase operon (SBM) and Methylmalonyl-CoA epimerase (MCE) - Succinyl-CoA is converted into Methylmalonyl-CoA-R by the methylmalonyl- CoA mutase ScpA. Methylmalonyl-CoA-R is converted into Methylmalonyl- CoA-S by MCE or an uncharacterised, native pathway. Methylmalonyl-CoA-S is converted into propionyl-CoA by the methylmalonyl-CoA carboxylase ScpB. The CoA from Propionyl-CoA is transferred onto Succinate from the citric acid cycle by the Propionyl-CoA: Succinate CoA transferase ScpC, resulting in the production of propionate and Succinyl-CoA.

• Determine whether E. coli  DH5 proposes SBM in genome and if it proposes any change that impact the resulting protein structure and function.
• Design and order DNA fragments from IDT to obtain Sleeping Beauty Mutase (SBM) which consists of genes ScpA, ScpB, ScpC, and
• Design and order the primers for amplifying SBM operon.

Figure 2 Illustrated diagrams of designed primers - A) The combined primers of Sbm Forward 1 and 2. Sbm Forward 1 annealed to ScpA at its 3’ end, and introduces a Ribosome binding site (RBS; green), AvrII restriction site (black), and half of Promoter BBa_J23110 (orange). Sbm Forward 2 annealed to the amplified sbm operon through containing a complete Promoter BBa_J23110 sequence, and introduced the remaining sequence, alongside XbaI (blue) and EcoRI (red) restriction sites. B) Illustrated diagram of Sbm Reverse Primer. Anneals to the end of ScpC (green), and introduces a SpeI restriction site (orange), and a NsiI restriction site (red).

Week 3 – Week 5 Amplify SBM and construct cloning vectors.

• Amplify the SBM operon with ordered primers as shown in Figure 1 by tow-step PCR. And analysed by gel electrophoresis.



Figure 3 Gel Electrophoresis of DNA from 2 PCR reactions - PCR-1 is PCR results from the amplification of the SBM operon from E. coli DH5-gDNA, with a band between 6kbp and 5kbp, and a band of below 500bp. PCR-2 used the products from PCR-1 as template DNA and used the Sbm forward 2 and Sbm Reverse primers – a single band of below 500bp can be observed.

• TA cloning with SBM to obtain more SBM operon without doing PCR many times.

SBM operon is ‘A-tailed’ prior to ligating into pCRTM2.1-TOPO, and transformed into One Shot® cells and plated. Following incubating overnight, blue colonies and white colonies could be observed. White colonies were used to purify plasmid DNA, and were analysed by digestion with EcoRI.



Figure 4. Analytical digest of TA colonies. Marker ladder used was NEB 1kb ladder, and all samples show a band between 3kbp and 4kbp, and a band below 500bp.

Bands of below 500bp could be observed, suggesting that some of the primer dimers may have been contaminating the extracted SBM operon sample, which were A-tailed, and effectively out- competing the much larger SBM operon fragments to be ligated into pCRTM2.1-TOPO.Again, another cloning method for the utilisation of the SBM operon had to be devised.

• Construct pSB3T5: MCE: Promoter- RFP and pSB3T5: Promoter-RFP followed by gel electrophoresis.



Figure 5 Restriction Analysis of various constructs. Marker ladder was Promega 1kb ladder. pSB3T5: MCE: Promoter-RFP was digested with SpeI and/or PstI. Single digests both produced bands of 4757bp, whereas double digest produced 2 bands of 887bp and 3870bp. pSB3T5 was digested with EcoRI and/or PstI. Single digestion results in bands of 4280bp, whereas double digest produced bands of 3211bp and 1069bp. pSB3T5: Promoter-RFP was digested with SpeI and/or PstI. Single digests both produced bands of 4156bp, whereas double digest produced 2 bands of about 3269bp, and 887bp. BBa_J23106 was digested with EcoRI and/or PstI. Single digestion results in bands of 2983bp, whereas double digest produced bands of 2038bp, and 945bp. PSB3T5: MCE was digested with EcoRI and/or PstI. Single digests both produced bands of 3845bp, whereas double digest produced 2 bands of 3211bp and 634bp.

• Insert the operon into BioBrick vevtor such as pSB3T5, through digesting with EcoRI and Nsi

Figure 6 a. Diagram illustrating the construction of pSB3T5: MCE. RFP (red) was cleaved from pSB3T5 by digestion with EcoRI and PstI. b. Diagram illustrating the construction of pSB3T5: Promoter-RFP. RFP (red) was excised from pSB3T5 by digesting EcoRI and PstI.

Week 6- Week7 Construct pSB3T5: SBM and pSB3T5: MCE: SBM

• Construct pSB3T5: SBM followed by Colony PCR. The strategy was showed in Figure 7 and gel electrophoresis of colony PCR was showed in Figure 8.



Figure 7 Diagram illustrating the construction of pSB3T5 - SBM. pSB3T5: Promoter- RFP was digested with SpeI and PstI, removing RFP (red). The SBM operon (orange) was digested with AvrII and NsiI. The overhangs generated are illustrated. The SBM operon was ligated into pSB3T5: Promoter downstream of the promoter (purple).

  

Figure 8 a. Image of DNA gel electrophoresis from colony PCR of transformants possessing pSB3T5: SBM using SBM Forward 1 and SBM Reverse Primers. Control lane is the same PCR with no template DNA. Control shows a large empty lane except for a single band below 250bp. All samples show a large smear that is intense from the well down to 5551bp, corresponding to the SBM operon, where it becomes less intense. b. Analytical digest of pSB3T5: SBM using XbaI and SpeI, followed by gel electrophoresis. Uncut sample showed 2 bands, one above 10kbp, and one between 8kbp – 6kbp. XbaI and SpeI single digests show a single band of 8790bp. XbaI and SpeI double digest shows two bands: a pSB3T5 backbone at 3250bp, and the SBM operon at 5540bp.

• Two different strategies to construct pSB3T5: MCE: SBM

Figure 9 a. Diagram illustrating the construction of pSB3T5: MCE: SBM. pSB3T5: MCE: Promoter-RFP was digested with SpeI and PstI, removing RFP (red). The SBM operon (orange) was digested with AvrII and NsiI. The overhangs generated are illustrated. The SBM operon was ligated into pSB3T5: Promoter downstream of the promoter (purple). MCE is indicated by black. b. Diagram illustrating the construction of pSB3T5: MCE: SBM. pSB3T5: SBM was digested with EcoRI and XbaI. pSB3T5: MCE was digested with EcoRI and SpeI to excise MCE (brown). The resulting overhangs are displayed. MCE was ligated into the plasmid upstream of the promoter (purple) and the SBM operon (orange), completing the plasmid.

Week 8 Propionate Assay

• Pellets of pSB3T5, pSB3T5: MCE, pSB3T5: SBM, and pSB3T5: MCE: SBM grown in LB broth were resuspended in M9 minimal media with 3% glucose and tetracycline in order to assay for the production of propionate.
• Detect the produced propionate by detecting the change in absorbance at 410nm using spectrophotometer.

Fe³⁺ ions have been shown to react with short chain fatty acids, such as propionate, where the ion complexes with the organic acid and is reduced. This changes the colour of the iron ion, and this change can be detected in a spectrophotometer by a change in absorbance at 410nm. (failed)

Table 1 Absorbance at 410nm of propionate standard solutions

Investigation of the effects of sucAB and sucCD on the adaption to propionic Acid and PHBV production (Siqi)

Week 1- Week 2 Amplify essential DNA fragments by ordered primers

• Design and order primers from IDT
• Amplify the sucAB and sucCD from coli DH5genome (Figure 1), sucAB and sucCD are expected to be 4 kb and 2kb respectively.

Figure 1 Gel electrophoresis image of sucAB and sucCD genes from PCR amplification on 1% agarose gel, contrast with 1KB ladder from NEB, generating fragments for the following experiments.

Week 3 – Week 5 Constructs establishment

• Construct plasmids: pSB3T5-sucAB, pSB3T5-sucCD, plasmid pSB3T5-X
• Transform recombinant plasmids to coli DH5
• Isolate the plasmids and double digest them with corresponding restriction enzymes for confirmation

Figure 2. A Gel electrophoresis image of pSB3T5-sucAB digested with EcoRI and HindIII, and run on 1% agarose gel, contrast with 1KB ladder from NEB. B.  Gel electrophoresis of pSB3T5-sucCD digested with EcoRI and HindIII, and run on 1% agarose gel, in the third well, there are two bands, the one located above is ringed DNAs are not be digested, sample loaded in this well is not cut completely.

Week 5 ---Week 7 Establish different E. coli trains

• Transform recombinant plasmids to coli forming strains SC1 to SC11. (shown in Table 1)

Table 1  Basic information of engineered E. coli strain

2. Pre-cultured strain SC1, SC2 and SC3 in LB medium
3. Inoculate the pre-culture to M9 minimal medium with 1% glucose, 0.01M propionic acid and 10M IPTG.
4. Optical density at 600nm was measured to plot the growth curve and growth rate was calculated using equation below.

               Table 2 Growth rate of E. coli strains SC1, SC2 and SC3

Figure 3 Growth curves of strain SC1, strain SC2 and strain SC3 that harboured plasmid pSB3T5-sucAB, plasmid pSB3T5-sucCD and plasmid pSB3T5-X respectively. 1% glucose, 0.01M propionate and 10 μM IPTG were added in the M9 medium.

Week 8 investigate the effect of sucAB and sucCD gene on growth and propionate adaption

• Pre-culture six different strains to Investigate the effect of sucAB and sucCD gene on growth
• Measure the germinate multiple* (GM) of each strain, which represented the proliferation capacity of cells (shown in figure 4)



Figure 4 Images of growth curves and Germinate Multiple - Stacked columns reflect growth rates of each strain under each case. Table at underneath the X-axis diaplays OD₆₀₀ measurement of six strains under serial propionate concentrations varying from 0 to 0.04 M at the interval of 0.01 M along with time line, time 0: immediately after strains are inoculated into M9 medium, 19: OD₆₀₀ was measured after cultivation of 19 hours, 24: OD₆₀₀ was measured after cultivation of 24 hours

• Determine the amount of propionic acid in the medium to investigate the effect of sucAB and sucCD gene on propionate uptake. Required standard curve was plotted during the pre-experimental. (shown in Figure 5)

Figure 5 Standard curve and the equation between the absorbance and propionate concentration.

• Obtain the amounts of propionic acid that utilized by coli strain SC7, SC8 and SC9.

Figure 6 The bar chart represented the amount of propionate taken by three different strains

*GM defined as final cell concentration / inoculation cell concentration   

Week 9 –Week 10 PHBV production and optimization

• Dry the cells after cultivation of 60 hours.
• Nile red plate staining to confirm the production of PHBV



Figure 7 Images of Nile red plate which are exposed to blue light or UV light; all six strains were spread on the plate for overnight culture 

• Measure the cell dry weight (CDW).



Figure 8 Absolute cell dry weight of each strain against propionate concentrations, indicating the yield of each strains - It can be seen that when 0.03 M, all three strains have largest absolute dry cell weight, since cultured enough time, the same as PHB production, which corresponding to the largest absorption of propionate in 0.03M

• Change the concentration of IPTG. The effect of different IPTG concentration on cell growth and PHBV production were compared



Figure 9 Growth of strain-SC7, SC8 and SC9 under different IPTG concentration, all the cases are cultured in the same condition. The line graph at left is the growth curve of strain-SC7 with IPTG concentration of 0, 0.05M and 0.1 M, the graph in the middle is from strain-SC8 and the line graph at right is strain-SC9.

• Measure the fluorescent intensity of three different strains using Plate Reader

Figure 10 Bar chart of Fluorescent intensity - Cells were cultured with different concentrations of IPTG, fluorescent intensity was measured at cultivation of 24 hours and 48 hours

Replace the T7 promoter with hybrid promoter, obtaining plasmids pSB3T5-sucAB-hypro and pSB3T5- sucCD-hypro

The Phasin and Hemolysin Secretion System (Owen)

Week 1 Design the strategy for plasmid construction 

• To investigate and optimized the level of Hemolysin transporter to PHB secretion, PCR strategy and digestion strategy were designed and ;utilised in plasmid construction.
• Order the primers



Figure 1 Diagram of new Biobricks development - The development of Lac promoter-Phasin-HlyA without stop codon through PCR strategy. DNA from the Lac promoter-Phasin-HlyA original Biobrick was used as a template to remove the stop codon in the end of Phasin sequence. The PCR product was then digested with DpnI (NEB) to remove the original DNA template then purified with QIAquick PCR Purification Kit (Qiagen), followed by self-ligation.



Figure 2 Diagram of new Biobricks development - The development of pSB3T5-T7-hlyDB-Pro-phaP-hlyA. Several Biobricks were used in this process for assembly, these included T7 promoter, Lac Promoter-PhaP-HlyA, HlyA-tag+Secretion system and pSB3T5-I52001. The purple lines represent the location of enzyme digestion. HlyBD and T7 promoter backbone was first obtained through digestion from their Biobricks then ligated together. The pSB1AK8 backbone of T7 promoter-HlyBD then was replaced by digestion strategy to form T7 promoter-HlyBD/pSB3T5. Lac promoter-Phasin-HlyA without stop codon were used as template to replace its promoter to J23100 promoter through PCT strategy. The parts of J23100 promoter-Phasin-HlyA and T7 promoter-HlyBD/pSB3T5 vector were then ligated together to form T7 promoter-HlyBD-J23100 promoter-Phasin-HlyA/pSB3T5.  



Figure 3 Diagram of new Biobricks development. The development of pSB3T5-T7-hlyDB-phaR-phaP-hlyA 

 

Week 2- Week 4 Constructs establishment 1 

1. Establish a new construct: DNA fragment pSB1AK8-T7-hlyBD (cloning strategy was shown in Figure 2) and transferred in E. coli BL21 (DE3)
2. Confirmation of successful pSB1AK8-T7-hlyBD plasmid with triple digestion
3. Establish a new construct: DNA fragment pSB3T5-T7-hlyBD (cloning strategy was shown in Figure 2) and transferred in E. coli BL21 (DE3)
4. Confirmation of successful pSB3T5-T7-hlyBD plasmid with double digestion



Figure 4 Agarose gel electrophoresis of restriction enzyme-digested Biobricks. A. pSB1AK8-T7-hlyBD plasmid (lane 1: 1 kb DNA ladder; 2: undigested plasmid; 3: EcoR1 digestion; 4: HindIII digestion; 5: PstI digestion; 6: EcoR1, HindIII and PstI triple digestion). B. pSB3T5-T7-hlyBD (lane 0 & 1: 1 kb DNA ladder; 2: undigested plasmid; 3: SpeI digestion; 4: PstI digestion; 5: SpeI and PstI double digestion)

 

Week 4 – Week 6.  Constructs establishment 2  

1. Stop codon removal from the original Biobrick (Lac promoter-Phasin-Hemolysin A/ pSB1C3, LPH/pSB1C3) with PCR strategy as shown in Figure 1 and transferred in E. coli BL21(DE3)
2. Confirmation of with Stop codon removed LPH/pSB1C3 via EcoRI and HindIII double digestion, which the HindIII restriction enzyme was not present in the original Biobrick and introduced through the PCR amplification
3. Establishment of two new constructs: pSB1C3-T7-hlyBD-Pro-phaP-hlyA and pSB1C3-T7-hlyBD-phaR-phaP-hlyA and transferred in E. coli BL21(DE3)
4. Confirmation of pSB1C3-T7-hlyBD-JPH and pSB1C3-RPH with EcoRI and PstI double digestion

 

Week 7 – Week 9 Cell culture for growth study  

1. Culture the E. coli BL21 (DE3), which harbouring the following plasmid(s) in M9 medium that contained 3% glucose with corresponding antibiotic(s) concentration, and assessed their optical density in different time points
   1. pSB1C3, LPH, LPH (without stop codon), pSB1C3 (Red fluorescent protein+), pSB1C3 (RFP-), pSB1C3, PHA operon, PHA operon + pSB1C3 (0, 3, 21, 25, 47, 51 and 71hours);   
   2. T7 promoter-HlyBD - JPH (0, 4, 7.5, 24, 28 and 94 hours); T7 promoter-HlyBD - JPH + PHA operon (0, 3, 20, 24 and 90 hours); in the case of IPTG induction, pSB3T5 (RFP+) and T7 promoter-HlyBD (0, 3.5, 4.5 and 72 hours), T7 promoter-HlyBD - JPH (0, 3.5, 5.5 and 71.5 hours), T7 promoter-HlyBD - JPH + PHA operon (0, 2.5, 19.5, 24.5, 42 and 47 hours), LPH (0, 3.5, 5.5, 71.5, 75.5, 78, 95, 100 and 117 hours), and LPH (without stop codon) (0, 3.5, 5.5, 17, 23 and 71.5 hours)
   3. phaCAB operon + pSB1C3 incubated in 50ml culture (250ml Flask) were measured at 0, 18, 24.5, 39.5, 43.5 and 63.5 hours)

 

   

   Figure 5 Agarose gel electrophoresis of Phasin-HlyA products. A. PCR product of Lac promoter-Phasin-HlyA with stop codon removal. B. Enzyme digestion of Lac promoter-Phasin-HlyA PCR product for stop codon removal (lane 1: 1 kb DNA ladder; 2: EcoRI and HindIII   double digestion) with the label of HlyA + pSB1C3 Backbone and Phasin. C. PCR product of pSB1C3-Lac promoter-Phasin-HlyA (stop codon -) with J23100 promoter forward and reverse primers 1; D. PCR product of pSB1C3-Lac promoter-Phasin-HlyA (stop codon -) with J23100 promoter forward and reverse primers 2; E. Enzyme digestion of constructed T7 promoter-HlyBD-JPH plasmid andT7 promoter-HlyBD-RPH plasmid (lane 1 & 4: 1 kd DNA ladder; 2-3: EcoRI and PstI double digestion for T7 promoter- HlyBD-JPH plasmid; 5-6 EcoRI and PstI double digestion for T7 promoter-HlyBD-RPH plasmid). 



Week 9 Determination of PHA production  

1. Nile red plate staining   



Figure 7 The growth study of E. coli Bl21 (DE3) strain with constructed HlyBD-Phasin-HlyA plasmid with and without IPTG (Triplicate). The results are represented as the mean OD600 ± S.E.M.



Figure 8 The growth study of PHA operon - The results are represented as the mean OD600 ± S.E.M. A. The growth study of E. coli BL21 (DE3) strain with pSB1C3, PHA operon, phaCAB operon+ pSB3T5 (RFP-) in 50ml tube (performed in triplicate). B. The growth study of E. coli BL21 (DE3) strain with phaCAB operon+pSB3T5 (RFP-) in 250ml Flask.  



Figure 9 The growth study of E. coli Bl21 (DE3) strain with pSB3T5-T7-hlyDB-phaP-hlyA and phaCAB operon with and without IPTG (Triplicate). The results are represented as the mean OD600 ± S.E.M.    

Week 9 – Week 10 Cell culture for PHA production 

1. Culture the E. coli BL21 (DE3) to investigate PHA production in different condition under M9 medium that contained 3% glucose with corresponding antibiotic(s) concentration
2. Measure fluorescent intensity of cultures to provide real-time information of PHA production. (semi-quantitative Nile red measurement) 

 Table 1 PHB production of recombinant E. coli BL21 (DE3) strain with PHA operon from 72 hours bacterial culture in M9 medium with 0.3% glucose (performed in singular). The results of OD600 are represented as the mean OD600 ± S.E.M. 

	Dry cell mass (g)	PHB production (g)	Melting Temperature (°C)	OD600
1 litre flask incubation (200ml culture sample)	0.59	0.008	165-170	2.244 ±  0.014
500ml flask incubation (100ml culture sample)	0.285	0.001	168-175	1.629 ±  0.035
250ml flask incubation (50ml culture sample)	0.17	Not measurable	160-169	1.913 ±  0.013

 
 

Table 2 The PHB production of E. coli BL21 (DE3) strain (PHA operon + pSB3T5) cell culture for 62 hours in M9 medium with 3% glucose (performed in duplicate)  

	Extracellular fraction			Intracellular fraction		PHB Production
	Amount of PHB in	CaCl2 added	Corrected amount of PHB	Amount of PHB	Melting Temperature
50ml culture sample X 2	0.016±0g	0.110g	0g	0.011±0g	165-170°C	0.11g/L
50 ml culture sample	0.0085±0g	0.055g	0g	0.006±0g	168-175°C	0.12g/L

 

Figure 10 Extracted product via PHB extraction protocol - A. Extracted product from intracellular fragment. B. Extracted product from extracellular fragment.    

 

Figure 11 Fluorescence intensity detection of E. coli BL21(DE3) strain with pSB1C3-T7-hlyBD-JPH and phaCAB operon under Nile red stain (performed in triplicate) - Results are represented as the mean fluorescent strength ± S.E.M. measured at 520 nm excitation and 590 nm emission wavelengths in 24 and 48 hours. Cultured in M9 medium with 3% glucose (performed in duplicate)