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B0064-porin281-Vib-ftl-porin194-Vib-folD

Sequence and Features

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

Due to the significant negative impact of pSEVA341 on bacterial growth, we aim to introduce the entire formate assimilation pathway solely through pSEVA321. However, the C1, C2, and C3 modules are too large to be placed on the same plasmid, otherwise Halomonas TD cannot accept them, or lead to serious growth impairments if they are accepted. Given that C1M can significantly enhance the ability of TD80 to assimilate formate, we have decided to integrate C1M into the TD80 genome first, and then express the C2 and C3 modules using pSEVA321.
Integrating C1M into the genome first requires replacing the inducible promoter with a constitutive one, and exploring which constitutive promoter is optimal. Previous fermentation data <a href="https://parts.igem.org/Part:BBa_K5226063">BBa_K5226063</a> indicated no clear trend in stepwise changes under IPTG concentration gradients, with even higher C1M expression observed when IPTG was set to zero. This may suggest leakage, implying that the expression intensity of C1M does not need to be excessively high. Consequently, we decided to replace the Mmp1 inducible promoter with two constitutive promoters, porin194 and porin281, which exhibit weaker expression intensities.
Meanwhile, 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 to conduct an orthogonal design of the promoter, hoping to achieve varied results.
Therefore, we ultimately decided to construct tuning plasmids with RBS B0064 and orthogonal design of the promoter as the experimental group, while creating control plasmids that only incorporate weak promoter tuning.

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 60-LB 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 50-MMF 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 60-LB 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 150-mL 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