Part:BBa_K5226078
321（C+cysNC）-194-4hbd-sucD-ogdA-194-orfZ
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2528
Illegal BamHI site found at 6605 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2981
Illegal NgoMIV site found at 3866
Illegal NgoMIV site found at 4977
Illegal NgoMIV site found at 5101
Illegal AgeI site found at 2037 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2103
Illegal SapI.rc site found at 2171
Illegal SapI.rc site found at 10221
Introduction
The existing methods for large-scale production of P34HB primarily rely on microbial fermentation. A key limiting factor in this process is the molar ratio of 4HB. Increasing the 4HB molar ratio can lead to a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in the polymer's deformation resistance. Therefore, enhancing the molar ratio of 4HB is crucial for the modification of P34HB.
Usage and Biology
After blocking the expression of cysNC in TD80, the strain is unable to synthesize methionine, making it difficult to grow in methionine-deficient media. We aim for only the recombinant TD80 containing the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster to thrive in 50 mM medium without the addition of antibiotics. To achieve this, we incorporated the cysNC gene into the pSEVA321 backbone, allowing TD80 transformed with the plasmid to synthesize methionine and grow in 50 mM medium lacking methionine.
Experimental characterisation
growth conditions
shake flask studies
experimental design
Post fermentation treatment
·Measurement of growth curve: 200 microliters of sample were taken at the fermentation time of 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 40, 48h, diluted to OD600 between 0.2~0.8, and measured with a spectrophotometer.
·Dry weight measurement: Take 15ml of bacterial solution, centrifuge evenly at 9000rpm for 5 minutes. Discard the supernatant, then add 15ml of water to resuspend the precipitate (2500rpm for 10 minutes). Centrifuge evenly for the second time, discard the supernatant, cover the tube with sealing film and puncture the hole. Place the sample in a -80 °C freezer for 2 days, then transfer it to a freeze dryer for 24 hours before weighing it.
·Content measurement: Take 30-40 mg of the sample into an esterification tube. Add 2 mL of esterification solution and 2 mL of chloroform to each tube. Heat the mixture at 99.9 °C for 4 hours, then cool it down. Add 1 mL of ultrapure water to each tube and mix well. Allow it to stand for 1 hour, then take 1 mL of the lower liquid from the filter head and analyze it using a gas chromatograph.
Data Processing and Analysis
By plotting the growth curves, it is evident that the growth of TD80 (△cysNC-key) is comparable to that of TD80 and TD80 (△cysNC-key) in the presence of 0.2 g/L Met. Consequently, cysNC is not an essential gene in the methionine synthesis pathway and cannot be used as a selective pressure to ensure continuous expression of the gene cluster in TD80.
After gas phase analysis, the molar ratio of P34HB produced by TD80 (△cysNC-key) & 321(C+cysNC)-194-4hbd-sucD-ogd-194-orfZ was significantly lower than that of TD80 & 321(C)-194-4hbd-sucD-ogd-194-orfZ. This suggests that some TD80 cells lost the pSEVA321 plasmid during fermentation and ceased to produce P34HB.
The dry weight results indicated that the growth of recombinant TD80 increased even without the addition of antibiotics. Therefore, identifying a new essential gene may be a viable strategy to enhance the 4HB mol% of P34HB, and this will be a focus of our future efforts.
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