Part:BBa_K5226075
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 1286
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 4902
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.
Prior to embarking on this project, our laboratory had already conducted research on the production of P34HB. It was found that the expression of the 4hbd-sucD-ogdA-orfZ gene cluster could increase the molar ratio of 4HB. Following fermentation using Mmp1 inducible promoter, the porin194 constitutive promoter was considered more suitable based on the concentration gradient induction trend observed with IPTG.
Since two plasmids, pSEVA321 and pSEVA341, are commonly used in the laboratory, the gene cluster has only been previously expressed through the pSEVA321 plasmid. Our intention is to introduce the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster into TD80 to synthesize P34HB, utilizing both the pSEVA341 and pSEVA321 plasmids, which allow us to evaluate which plasmid yields better results.
Usage and Biology
Although TD80 possesses its own phaAB gene cluster for synthesizing 3HB-CoA, it is almost incapable of synthesizing 4HB-CoA. To address this, a heterologous gene cluster comprising ogdA, sucD, 4hbd, and orfZ has been introduced to facilitate the synthesis of 4HB-CoA. Subsequently, the endogenous phaC gene catalyzes the conversion of both 3HB-CoA and 4HB-CoA into P34HB.
Experimental characterisation
growth conditions
shake flask studies
experimental design
Post fermentation treatment
·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
The experimental results indicated that the 4HB molar ratio achieved by introducing the pSEVA321 was higher than that of the pSEVA341. However, the dry weight decreased somewhat compared to the wild TD80.
In order to stabilize the cell dry weight while increasing the 4HB molar ratio, we decided to explore the effect of the pSEVA321 on cell dry weight and make improvements. For further experiments, please turn to <a href="https://parts.igem.org/Part:BBa_K5226077">BBa_K5226077</a>.
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