Week 1- Week 2 Primers design and experimental preparation
Primers for changing gene order in pha operon are designed in Gibson Assembly website
Week 3- Week 5 Building and confirming constructs
Due to the special primer design from Gibson, except backbone fragment, all of the other fragments were amplified by primers with overhang. In this case, a problem was caused by wrong identity of annealing sequence and overlap sequence. Therefore, it is necessary to produce fragments without overlap firstly, and used them as template, in order to avoid the impact of the overlap and annealing sequence.
Week 6- Week 8
Figure 1. Results of colony PCR. The fragments of 4kb size were expected to be the positive results (a)BCA are 12 colonies from plates expressed by phaBAC plasmid. (b)BCA are 12 colonies from phaBCA plasmid. (c) represents 12 colonies from phaABC expression plasmid. 11 colonies in (d) are from phaCBA plasmid. (e) is the result if negative control of this PCR experiment, which represents colonies from expression of empty vector. (f) shows 8 colonies from phaACB plasmid. Finally, (g) is the second negative control of purity water used in this reaction, no band means dH2O used is pure.
Week 9- Week 10 PHB production and extraction
Figure 2 Nile red plates images of E. coli that harboured new constructs. On each plate, half plate is spread by negative control- pSB1C3 empty vector, which should not have any fluorescence, another half is plated with new operon plasmids culture.
Table 1 OD value detected by Nile Red plate reader
Hours
BAC
BCA
CBA
ACB
ABC
24 h
2% Glucose
0.45±0.02
0.48±0.01
0.65±0.01
0.69±0.01
0.42±0.01
3% Glucose
0.44±0.01
0.64±0.01
0.63±0.01
0.47±0.01
45 h
0.66±0.01
0.70±0.01
0.94±0.02
0.99±0.02
0.61±0.01
0.68±0.02
0.94±0.01
0.98±0.03
0.68±0.01
Table 1 is the OD600 value of Nile Red plate reader, culture fed with 2% glucose and 3% glucose are incubated for 24h and 45h, then stained by Nile Red, and placed into a black 96 well microplate and read by Tecan Infinate M200 (provided by Edinburgh Genome Foundry). Excitation wavelength is 520 nm, and emission wavelength is 590 nm. Data are average number ± SEM (standard error of mean), n=3. P-value<0.05.
Table 2 Fluorescence intensity measurement by Nile red plate reader
1,683±132
2,371±90
35,876±850
41,757±586
4,534±311
1,758±48
2,437±108
40,330±134
31,486±670
4,651±222
5,374±97
5,432±236
29,874±685
42,295±793
5,054±182
5,415±270
5,164±41
27,890±989
28,311±1988
4,499±95
Table 2 shows the fluorescence value detected by Nile red plate reader, samples stained by Nile Red, and placed into a black 96 well microplate and read by Tecan Infinate M200 (provided by Edinburgh Genome Foundry). Excitation wavelength is 520 nm, and emission wavelength is 590 nm. Data are average number ± SEM (standard error of mean), n=3, P-value<0.05.
The Production of PHB
Figure 3 Image of extracted PHB after cultivation of 45 hours - Figure 3 indicates the PHB products extracted from E. coli BL21 harboring six pha operons and empty vector plasmid, samples shown in figure 3 were incubated for 45h
Figure 4 Image of extracted PHB after cultivation of 72 hours - Figure 4 shows the PHB products from E. coli BL21 harboring phaCBA and phaACB, which incubated for 72h
Table 3 Dry weight of produced PHB and melting temperature
Operon construct
Dry weight
Melting temperature start
Finish
Yield of PHB after 45h cultivation
Empty vector
~0
/
phaCAB origin order
0.237 g
150
185
phaACB
0.201 g
156
180
phaCBA
0.072 g
160
186
phaBCA
0.021 g
158
184
Yield of PHB after 72h cultivation
0.044 g
148
165
0.18 g
176
Table 3 indicates the dry weight and melting temperature of all PHB products extracted in this project
Week 1- Week 2 Designing the fragments and primers
Week 3- Week 5 constructs establishment and culture condition optimization
Figure 1. B1 and B2 fragments were codon-optimised for E. coli and synthesised by IDT. The 696 bp overlap was in green and restriction enzyme sites were coloured by red. Two pairs of primers were ordered from IDT to amplify bktB, among which B1 forward primer and B2 reverse primer were used to amplify bktB fragments.
Three concentrations (8mM, 32mM and 48mM) were tested using E. coli cells that harboured pSB1C3-phaCAB.
Figure 2. Plasmid of control plasmid pSB1C3-RFP. The new control plasmid pSB1C3 was obtained by digesting pSB1C3-RFP with restriction enzyme: NotI followed by self-ligation.
Figure 3. The bktB fragments and psB1C3-phaCB-bktB fragments were highlighted by red squares.
Week 6 – Week 7. Nile Red quantitative measurement
The transformation was tested by double digestion and colony PCR
Figure 4. Nile red staining plate was exposed to the blue light and UV light respectively. High fluorescent was observed on the right part (cells containing pSB1C3-phaCAB plasmid) due to the produced PHA.
Week 8 – week 9 cell culture and PHA production
Figure 5. Samples were collected at different cultivation times and stained by Nile red fluorescent dye. The fluorescent intensity increased during the cultivation times.
Week 9. Determination of PHA production
Figure 6. Nile red Plate staining of cells harbouring pSB1C3-phaCAB-bktB or pSB1C3-phaCB-bktB. Compared with control cells harbouring control plasmid, the production of PHA was confirmed.
OD600
Volume (ml)
Total PHA (g)
PHA (mg/ml)
pSB1C3
1.35
100ml
0
pSB1C3-phaCAB
1.707
0.044
0.44
pSB1C3-phaCB-bktB
2.15
0.023
0.23
pSB1C3-phaCAB-bktB
1.875
0.021
0.21
Melting temperature of extracted PHA was measured and compared with PHB and PHBV products which were brought from Sigma. Tm of standard pure PHB product was between 170 °C and 180 °C and Tm of PHBV (12 % 3HV) was between 160 °C and 164 °C.The melting temperature of extracted PHA differed from different constructs. The PHA extracted from cell harbouring pSB1C3-phaCAB plasmids showed higher melting temperature with approximately 165 °C. While the PHA extracted from the cells harbouring pSB1C3-phaCB-bktB or pSB1C3-phaCAB-bktB plasmids presented lower melting temperature at range of 150 °C -155 °C and 158 °C -162 °C respectively.
Week 1 Experimental preparation
Table 1 Basic information of BioBricks
Biobrick
Well
Plate
Insert Length
Backbone
Antibiotics Resistance
BBa_K390501
19H
6
1136 bp
chloramphenicol
BBa_K1149051
12M
4
4271 bp
Week 2 Designing the fragments and primers
phaR promoter-phaR-phaP promoter (proR-phaR-proP) and primers for amplifying DNA fragment of phaP and HlyA
Figure1. Strategy of R-P-H assembly
Week 3 –Week 5 Constructs establishment
Figure 2 Images of 1% gel electrophoresis - A.1.0% agarose gel electrophoresis for PCR product of phaP and phaP-HlyA. Lane: 1. 1 kb ladder (molecular weight marker, NEB). 2. 25μL PCR reaction for phaP (569 bp). 3. 25μL PCR reaction for phaP-HlyA (759 bp). B. 1.0% agarose gel electrophoresis for PCR product of proR-phaR-proP. Lane: 1. 100 bp ladder (NEB). 2. 25μL reaction of PCR amplification for proR-phaR-proP (853 bp).
Figure 3 Image of diagnostic digest of pSB3T5-phaP and pSB3T5-phaP-hlyA with NsiI and PstI. Lanes - 1. 1 kb ladder (molecular weight marker, NEB). 2 and 3. Potential pSB3T5-phaP digested with NsiI and PstI. 4 and 5. Potential pSB3T5-phaP-HlyA digested with NsiI and PstI.
Week 6 Repeat the experiments and finish constructs establishment.
Figure 4 Images of gel electrophoresis. a. Diognostic digest of R-P and R-P-HlyA with EcoRI and PstI. Lanes: 1. 1 kb ladder (molecular weight marker, NEB). 2. Potential R-P digested with EcoRI and PstI. 3. Potential R-P-H digested with EcoRI and PstI. b. 1.0% Agarose gel electrophoresis for PCR product of plasmid pSB3T5-phaP-HlyA (del) and R-P-HlyA (del).Lane: 1. 1kb ladder (NEB). 2. 25μL reaction of PCR amplification pSB3T5-phaP-HlyA. 3. 25μL reaction of PCR amplification R-P-HlyA.
Repeat the co-transformation of phaCAB operon and R-P with lower concentration of antibiotics (8 μg/ml tetracycline and 20 μg/ml chloramphenicol).
Transform plasmid R-P into E. coli BL21 competent cells.
Transform the ligation of pSB3T5 without RFP (RFP (-) into E. coli DH5α competent cells.
Figure 5 Image of Agarose gel electrophoresis. a. 1.0% Agarose gel electrophoresis of colony PCR product for screening the positive construct of P. Lane: 1. 1kb ladder (Promega). 2-14. 25μL reaction of colony PCR amplification for proP-phaP (bp) with different colony extraction. The positive construct was expected to show a band with size of 1067 bp (proP-phaP + extra sequence in between the VF and VR primers.). b. 1.0% Agarose gel electrophoresis of colony PCR product for screening the positive construct of R.
Week 8 Transformation and colony PCR
Co-transform the phaCAB operon and R-P-HlyA (del) into E. coli BL21 competent cells
Co-transform the phaCAB operon and P into E. coli BL21 competent cells.
Transform the R-P-HlyA (del) into E. coli BL21 competent cells.
Transform the P into E. coli BL21 competent cells.
Figure 6 Image of 1.0% Agarose gel electrophoresis of colony PCR product for screening the positive construct of R - Lane: 1. 1 kb ladder (Promega). 2-14. 25μL reaction of colony PCR amplification for proR-phaR with different colony extraction.
The positive construct was expected to show a band with size of 935 bp (proR-phaR + extra sequence in between the VF and VR primers.
Week 9—Week 10. Culture bacteria to produce PHB
Figure 7 Nile red plates to confirm PHB production (48 hours)
In each picture of a, b, c, d, e, there were 4 Nile red plates. The left plates in the first rows were strains harbouring phaCAB operon and constructs. The right plates in the first row were strains containing the construct only. The left plates in the second row were strains harbouring phaCAB operon only. The right plates in the second row were pSB1C3 and pSB3T5 backbones. phaCAB operon strain showed strong signal of fluorescence because of PHB produced and the backbone strain did not due to no PHB produced.
Figure 7 The growth curves of E. coli strains that harboured different recombinant constructs
Table 2 PHB production
Intracellular PHB
Secreted PHB
Mass of CaCl2
phaCAB operon+pSB3T5-R-P
0.071 g
0.057 g
0.05549 g
0.0015 g
phaCAB operon+pSB3T5-R-P-H
0.043 g
0.086 g
0.0305 g
phaCAB operon+pSB3T5-R
0.065 g
0.058 g
0.0020 g
phaCAB operon +pSB3T5-P
0.031 g
0.030 g
0.0000 g
phaCAB operon +pSB3T5
0.010 g
0.038 g
pSB1C3+pSB3T5
0.000 g
0.042 g
Figure 8 Column graph for dry weight of secreted and intracellular PHB among different stains
Table 3 The melting temperature of produced PHB
Strains
Melting Temperature
156-164°C
160-168°C
phaCAB operon +pSB3T5-R
152-164°C
phaCAB operon+pSB3T5
162-170°C
\
Week 1 – Week 2
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.
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.
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.
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.
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.
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
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.
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
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
Sample
Absorbance at 410nm (arbitrary units)
0 g/l propionate
0.5 g/l propionate
0.017
1.0 g/l propionate
0.003
1.5 g/l propionate
-0.008
2.0 g/l propionate
2.5 g/l propionate
0.002
3.0 g/l propionate
0.024
3.5 g/l propionate
0.026
4.0 g/l propionate
-0.003
psb3T5
-0.5
psb3T5: MCE
-0.236
psb3T5: SBM
-0.232
psb3T5: MCE: SBM
-0.288
Week 1- Week 2 Amplify essential DNA fragments by ordered primers
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
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
Table 1 Basic information of engineered E. coli strain
Bacterial strains:
Relevant information
Escherichia coli SC1
Escherichia coli SC2
Escherichia coli SC3
Escherichia coli SC4
Escherichia coli SC5
Escherichia coli SC6
Escherichia coli SC7
Escherichia coli SC8
Escherichia coli SC9
Escherichia coli SC10
Escherichia coli SC11
Escherichia coli BL21 containing pSB3T5-sucAB
Escherichia coli BL21 containing pSB3T5-sucCD
Escherichia coli BL21 containing pSB3T5-X
BL21 containing pSB3T5-sucAB and pSB1C3-PHA
BL21 containing pSB3T5-sucCD and pSB1C3-PHA
BL21 containing pSB3T5-X and pSB1C3-PHA
BL21 containing pSB3T5-AB and empty pSB1C3
BL21 containing pSB3T5-CD empty pSB1C3
BL21 containing pSB3T5-X empty pSB1C3
BL21 containing pSB3T5-sucAB-hypro and pSB1C3-PHA
BL21 containing pSB3T5-sucCD-hypor and pSB1C3-PHA
Table 2 Growth rate of E. coli strains SC1, SC2 and SC3
2
3
5
10
60
72
SC1
0.18
0.503667
0.936667
1.101
1.195333
1.379667
1.3666
SC2
0.489333
1.034
1.238
1.365
1.440667
1.36588
1.36522
SC3
0.28
0.828
1.497333
1.618333
1.755
1.94
1.9
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
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
Figure 5 Standard curve and the equation between the absorbance and propionate concentration.
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
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
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
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.
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
Week 1 Design the strategy for plasmid construction
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
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
Week 7 – Week 9 Cell culture for growth study
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
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
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)
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
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
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)
