Difference between revisions of "Part:BBa K5226074"
 
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One of the goals of iGEM24-SCUT-China-A is to use synthetic biology tools to obtain <i>Halomonas</i> TD strains that can metabolize formate. We chose to <b>introduce the formate assimilation pathway</b> to enable it to utilize formate, a difficult-to-recover product in CDE.
 
One of the goals of iGEM24-SCUT-China-A is to use synthetic biology tools to obtain <i>Halomonas</i> TD strains that can metabolize formate. We chose to <b>introduce the formate assimilation pathway</b> to enable it to utilize formate, a difficult-to-recover product in CDE.
 
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For the first method, <b>based on previous studies obtained from literature research</b>,[1][2][3][4]we selected the tetrahydrofolate (THF) cycle imported from <i>Methylobacterium extorquens</i>  AM1 and strengthened the endogenous C2 and C3 modules of <i>Halomonas</i>  TD.
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For the first method, <b>based on previous studies obtained from literature research</b>,[1][2][3][4]we selected the tetrahydrofolate (THF) cycle imported from <i>Methylobacterium extorquens</i>  AM1.
 
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As a second approach, <b>based on the homology between <i>Vibrio natriegens</i> and <i>Halomonas</i> TD </b>[5], we chose to import the C1, C2, and C3 modules from <i>Vibrio natriegens</i>.  
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As a second approach, <b>based on the homology between <i>Vibrio natriegens</i> and <i>Halomonas</i> TD </b>[5], we chose to import the C1 module from <i>Vibrio natriegens</i>.  
 
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===Usage and Biology===
 
===Usage and Biology===
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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 <i>Halomonas</i> 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 <b>integrate C1M into the TD80 genome first, and then express the C2 and C3 modules using pSEVA321.</b>
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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.
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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 <b>no clear trend in stepwise changes under IPTG concentration gradients</b>, 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 <b>replace the Mmp1 inducible promoter with two constitutive promoters, porin194 and porin281, which exhibit weaker expression intensities</b>.  
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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, <b>while adjusting the promoter strength, we decided to change the RBS to the moderately intense B0064 for optimization</b>.Additionally, we <b>divided the C1M gene into two segments to conduct an orthogonal design of the promoter</b>, hoping to achieve varied results.
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Previous fermentation data indicated <b>no clear trend in stepwise changes under IPTG concentration gradients</b>. In fact, even at an IPTG concentration of 0, the expression intensity was higher. Therefore, we have decided to <b>replace the Mmp1 inducible promoter with two constitutive promoters, porin194 and porin281, which exhibit weaker expression intensities.</b>
 
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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.  
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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 <b>change the RBS to the moderately intense B0064 for optimization</b>. Additionally, we <b>divided the C1M gene into two segments for tuning</b>, hoping to achieve varied results.
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<br>
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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.
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<html> <img src="https://static.igem.wiki/teams/5226/parts/design-of-tuning.png" width="700px">
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==Experimental characterisation==
 
==Experimental characterisation==
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<h3>growth conditions</h3>
 
<h3>growth conditions</h3>
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<p>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.
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<html> <img src="https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-2.jpg" width="700px">
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<h3>shake flask studies</h3>
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<p>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.
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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.
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<h3>shake flask studies</h3>
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<html> <img src="https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-4.jpg" width="700px">
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<h3>experimental design</h3>
 
<h3>experimental design</h3>
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<b>Experimental group:</b>
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Using 15g/L sodium formate as the sole carbon source
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Adding 25 μ g mL-1 chlorocatechol
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1.Recombinant TD80 with tuning plasmid B0064 and promoter for orthogonal design
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2.Recombinant TD80 with weaker promoter only
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<b>——Compare and determine the most effective combination</b>
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<b>Control group:</b>
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1. Wild TD80 without carbon source, using only 50MM as culture medium:
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eliminating the influence of other factors on TD80 growth
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2. Wild TD80 containing 15g/L sodium formate:
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observe the effect of introducing C1M on TD80 assimilation of formate;
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<h3>Post fermentation treatment</h3>
 
<h3>Post fermentation treatment</h3>
  
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Considering liquid chromatography as a <b>quantitative analysis</b>, 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 <b>B0064-Vib-281-ftl-194-folD</b> was selected as the most effective.   
 
Considering liquid chromatography as a <b>quantitative analysis</b>, 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 <b>B0064-Vib-281-ftl-194-folD</b> was selected as the most effective.   
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<br>Next, we plan to integrate the most effective modules into the genome. For details, please refer to <a href="https://parts.igem.org/Part:BBa_K5226077">BBa_K5226077</a>.  
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Given our hope that <i>Halomonas</i> 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, <b>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.<br>
  
  

Latest revision as of 03:16, 1 October 2024


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

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1189
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1189
    Illegal NheI site found at 2375
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1189
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1189
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1189
    Illegal NgoMIV site found at 455
  • 1000
    COMPATIBLE 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.