Part:BBa_K5226061
Mmp1-Vib-C1M
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 and strengthened the endogenous C2 and C3 modules of Halomonas TD.
As a second approach, based on the homology between Vibrio natriegens and Halomonas TD [5], we chose to import the C1, C2, and C3 modules 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
E. coli was cultured at 37 °C in an LB medium containing (g L−1) 10 NaCl, 10 tryptone, and 5 yeast extract. H. bluephagenesis was cultured at 37 °C in a 60LB medium, namely, the LB medium supplemented with 60 g L−1 NaCl. A 20-LB medium indicates the LB medium containing 20 g L−1 NaCl. Moreover, 15 g L−1 agar was added before autoclaving for preparing solid media in Petric plates. Ampicillin (100 μg mL−1), chloramphenicol (25 μg mL−1), kanamycin (50 μg mL−1), or spectinomycin (100 μg mL−1) were added to the above media whenever necessary.
shake flask studies
H. bluephagenesis TD80 and its derivatives were cultivated in 50MMF medium in shake flask studies. The 50MM medium was composed of (g/L): NaCl 50, sodium formate 15, yeast extract 1, CO(NH2)2 0.25, MgSO4 0.2, Na2HPO4·12H2O 9.65, KH2PO4 1.5, trace element solution I 10 mL/L and trace element solution II 1 mL/L. The composition of trace element solution I was (g/L): Fe(III)-NH4-citrate 5, CaCl2 2, HCl 1 M. The trace element solution II was composed of (mg/L): ZnSO4·7H2O 100, MnCl2·4H2O 30, H3BO3 300, CoCl2·6H2O 200, CuSO4·5H2O 10, NiCl2·6H2O 20 and NaMoO4·2H2O 30. The pH-value of the medium was adjusted to approximately 9.0 using 5 M NaOH.
the microbial glycerol stocks were resuscitated by streaking on fresh plates. Then constructed plasmid was transferred into Halomonas TD80 through modified conjugation method using E.coli S17-1 as donor cells. Single colonies from newly-conjugated plates were picked and inoculated in the 60LB liquid medium for 12 h at 200 rpm to acquire the first seed culture, which was further grown on a fresh 60-LB liquid medium at a volume ratio of 1%. The second seed culture was inoculated for 12 h at 200 rpm. Afterward, it was inoculated into 150mL conical flasks containing 20 mL of the defined minimal medium at a volume ratio of 5% and cultivated for 48 h at 200 rpm. Antibiotics were added if needed. The temperature for all cultivations was 37 °C.
experimental design
Experimental group:Using 15g/L sodium formate as the sole carbon source
Set IPTG induction concentration gradients of 0, 2, 5, and 10mg/L
1. Importing the Vib C1M plasmid into TD80:
Observing the effect of Vibrio natriegens' C1M on enhancing the formate assimilation ability of TD80
2. Importing the AM1 C1M plasmid into TD80:
Observing the effect of Methylobacterium extorquens AM1 C1M on enhancing the formate assimilation ability of TD80
Control group:
1. Wild TD80 without carbon source, using only 50MM as culture medium:
eliminating the influence of other factors on TD80 growth
2. Wild TD80 containing 15g/L sodium formate as the sole carbon source:
observe the effect of introducing C1M on TD80 assimilation of formate;
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% After importing C1M, the ability of TD80 to assimilate formate was significantly improved. We urgently hope to explore whether overexpression of C2M and C3M will further enhance the ability of TD80 to assimilate formate. For further experiments, please turn to BBa_K5226063.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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