Part:BBa_K5226061
Mmp1-Vib-C1M
Contents
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1199
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1199
Illegal NheI site found at 2161 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1199
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1199
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1199
Illegal NgoMIV site found at 465 - 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
The construct referred to as C1M is part of the formate assimilation pathway and consists of three key enzymes: formate-tetrahydrofolate ligase, methenyltetrahydrofolate cyclohydrolase, and methylenetetrahydrofolate dehydrogenase. Together, these enzymes convert formate into methylene tetrahydrofolate.
Since the C1M module is absent in Halomonas TD , we selected the Mmp1 inducible promoter for our experiments. We induced this promoter with a series of IPTG concentrations during fermentation to identify the optimal expression intensity for C1M.
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.
Data Processing and Analysis
The experimental results showed that the introduction of AM1-C1M increased the growth of TD80 by 67.33% under the culture conditions with sodium formate as the sole carbon source, while the introduction of Vib-C1M increased it by 59.67%.
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.
For further experiments, please refer to <a href="https://parts.igem.org/Part:BBa_K5226074">BBa_K5226074</a> for further examination.
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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