Part:BBa_K5226074
B0064-porin281-Vib-ftl-porin194-Vib-folD
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1189
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1189
Illegal NheI site found at 2375 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1189
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1189
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1189
Illegal NgoMIV site found at 455 - 1000COMPATIBLE WITH RFC[1000]
Introduction
One of the goals of iGEM24-SCUT-China-A is to use synthetic biology tools to obtain Halomonas TD strains that can metabolize formate. We chose to introduce the formate assimilation pathway to enable it to utilize formate, a difficult-to-recover product in CDE.
For the first method, based on previous studies obtained from literature research,[1][2][3][4]we selected the tetrahydrofolate (THF) cycle imported from Methylobacterium extorquens AM1.
As a second approach, based on the homology between Vibrio natriegens and Halomonas TD [5], we chose to import the C1 module from Vibrio natriegens.
Usage and Biology
Given that the Mmp1 promoter requires IPTG for induction, which can be costly, and considering the challenges posed by concentration gradients in large industrial fermenters—where it is difficult to ensure uniform IPTG distribution to all bacteria—we propose converting the inducible promoter into a constitutive promoter.
Previous fermentation data indicated no clear trend in stepwise changes under IPTG concentration gradients. In fact, even at an IPTG concentration of 0, the expression intensity was higher. Therefore, we have decided to replace the Mmp1 inducible promoter with two constitutive promoters, porin194 and porin281, which exhibit weaker expression intensities.
After discussing this with our teacher, we considered that RBS2000 had previously been used as the ribosome binding site for C1M, and it yielded the highest expression intensity among the series of RBSs. Therefore, it would not be meaningful to focus solely on weakening the promoter at this stage. Instead, while adjusting the promoter strength, we decided to change the RBS to the moderately intense B0064 for optimization. Additionally, we divided the C1M gene into two segments for tuning, hoping to achieve varied results.
Based on our own insights and our teacher's advice, we ultimately decided to construct tuning plasmids with RBS B0064 and promoter tuning as the experimental group, while creating control plasmids that only incorporate weak promoter tuning.
Experimental characterisation
growth conditions
shake flask studies
experimental design
Post fermentation treatment
To ensure the measurement accuracy of the spectrophotometer, we diluted the bacterial solution 5 times and measured OD600.
To accurately measure the ability of TD80 to assimilate formate, we diluted the bacterial solution tenfold and centrifuged the filter head. Subsequently, we used liquid chromatograph to measure the residual concentration of sodium formate in the bacterial solution.
Data Processing and Analysis
Analysis of the data revealed that the B0064-Vib-281-ftl-194-folD group had the lowest residual sodium formate concentration, which was 15.3% lower than that of the wild TD80 group. However, regarding OD600 measurements, B0064-AM1-194- ftfl-281-fch-mtdA exhibited the best growth, showing a 66.5% increase compared to wild TD80.Considering liquid chromatography as a quantitative analysis, the amount of formate assimilated by TD80 was accurately determined. Furthermore, the difference in OD600 between the two groups was not significant. Finally, the combination of B0064-Vib-281-ftl-194-folD was selected as the most effective.
Given our hope that Halomonas TD can utilize CO2 electrochemical derivatives instead of formate as their sole carbon source for growth, we initially discovered that the THF system effectively supports Halomonas TD using formate as a single carbon source. Building on this, we began to explore whether the THF system could facilitate dual carbon source fermentation by primarily using acetate, while allowing TD80 to absorb and utilize formate.
References
[1] Kim S, Lindner S N, Aslan S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature chemical biology, 2020, 16(5): 538-545.
[2] Yishai O, Bouzon M, Doring V, et al. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli[J]. ACS synthetic biology, 2018, 7(9): 2023-2028.
[3] Turlin J, Dronsella B, De Maria A, et al. Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation[J]. Metabolic Engineering, 2022, 74: 191-205.
[4] Claassens N J, Bordanaba-Florit G, Cotton C A R, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator[J]. Metabolic Engineering, 2020, 62: 30-41.
[5] Tian J, Deng W, Zhang Z, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.
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