Difference between revisions of "Part:BBa K5226074"

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	<partinfo>BBa_K5226074 SequenceAndFeatures</partinfo>		<partinfo>BBa_K5226074 SequenceAndFeatures</partinfo>
−	==Introduction==	+	<h2>Introduction</h2>
−	The existing methods for large-scale production of P34HB primarily rely on microbial fermentation. A key limiting factor in this process is the molar ratio of 4HB. Increasing the 4HB molar ratio can lead to a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in the polymer's deformation resistance. Therefore, enhancing the molar ratio of 4HB is crucial for the modification of P34HB.	+	<p>
−		+	<br>
−	Prior to embarking on this project, our laboratory had already conducted research on the production of P34HB. It was found that the expression of the 4hbd-sucD-ogdA-orfZ gene cluster could increase the molar ratio of 4HB. Following fermentation using Mmp1 inducible promoter, the porin194 constitutive promoter was considered more suitable based on the concentration gradient induction trend observed with IPTG.	+	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.
−		+	<br>
−	Since two plasmids, pSEVA321 and pSEVA341, are commonly used in the laboratory, the gene cluster has only been previously expressed through the pSEVA321 plasmid. Our intention is to introduce the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster into TD80 to synthesize P34HB, utilizing both the pSEVA341 and pSEVA321 plasmids, which allow us to evaluate which plasmid yields better results.	+	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.
		+	<br>
		+	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>.
		+	<br>
		+	<br>
	===Usage and Biology===		===Usage and Biology===
−	Although TD80 possesses its own phaAB gene cluster for synthesizing 3HB-CoA, it is almost incapable of synthesizing 4HB-CoA. To address this, a heterologous gene cluster comprising ogdA, sucD, 4hbd, and orfZ has been introduced to facilitate the synthesis of 4HB-CoA. Subsequently, the endogenous phaC gene catalyzes the conversion of both 3HB-CoA and 4HB-CoA into P34HB.	+	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.
		+	<br>
		+	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>
		+	<br>
		+	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.
		+	<br>
		+	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.
		+	<br>
		+	<html> <img src="https://static.igem.wiki/teams/5226/parts/design-of-tuning.png" width="700px">
		+	</html>
	==Experimental characterisation==		==Experimental characterisation==
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	<body>		<body>
	<h3>growth conditions</h3>		<h3>growth conditions</h3>
−	<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.	+	<html> <img src="https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-2.jpg" width="700px">
−	<html>	+	</html>
−	<body>	+
−	<h3>shake flask studies</h3>	+
−	<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.	+
	<br>		<br>
	<br>		<br>
−	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[6]. 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.	+	<html> <img src="https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-3.jpg" width="700px">
		+	</html>
		+
		+	<h3>shake flask studies</h3>
		+	<html> <img src="https://static.igem.wiki/teams/5226/parts/bba-k5226060-mmp1-am1-c1m-4.jpg" width="700px">
		+	</html>
		+
		+	<h3>experimental design</h3>
		+	<html> <img src="https://static.igem.wiki/teams/5226/parts/experiment-design-of-tuning.png" width="700px">
		+	</html>
		+	<h3>Post fermentation treatment</h3>
		+
		+	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.
		+	<br>
	<html>		<html>
	<body>		<body>
−	<h3>experimental design</h3>	+	<h3>Data Processing and Analysis</h3>
−	Using 35g/L sodium acetate as the sole carbon source	+	Analysis of the data revealed that the <b>B0064-Vib-281-ftl-194-folD</b> group had the <b>lowest residual sodium formate concentration</b>, which was <b>15.3%</b> lower than that of the wild TD80 group. However, regarding <b>OD600 measurements</b>, <b>B0064-AM1-194- ftfl-281-fch-mtdA</b> exhibited the best growth, showing a <b>66.5%</b> increase compared to wild TD80.
	<br>		<br>
		+	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.
	<br>		<br>
−	Experimental group:	+	<html> <img src="https://static.igem.wiki/teams/5226/parts/result-of-tuning1.png" width="700px">
−	Importing pSEVA321 & pSEVA341 into TD80:	+	</html>
−	Compare the effects of introducing the same gene cluster into TD80 using different vectors, focusing on factors such as the 4HB molar ratio, dry weight, yield, and other relevant metrics.	+
	<br>		<br>
	<br>		<br>
−	Control group:	+	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>
−	Wild TD80 containing 35g/L sodium acetate as the sole carbon source:	+
−	Assess the impact of introducing gene clusters	+
−	<html>	+
−	<body>	+	<h2>References</h2>
−	<h3>Post fermentation treatment</h3>	+	[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.
−	·Dry weight measurement: Take 15ml of bacterial solution, centrifuge evenly at 9000rpm for 5 minutes. Discard the supernatant, then add 15ml of water to resuspend the precipitate (2500rpm for 10 minutes). Centrifuge evenly for the second time, discard the supernatant, cover the tube with sealing film and puncture the hole. Place the sample in a -80 °C freezer for 2 days, then transfer it to a freeze dryer for 24 hours before weighing it.	+
	<br>		<br>
−	·Content measurement: Take 30-40 mg of the sample into an esterification tube. Add 2 mL of esterification solution and 2 mL of chloroform to each tube. Heat the mixture at 99.9 °C for 4 hours, then cool it down. Add 1 mL of ultrapure water to each tube and mix well. Allow it to stand for 1 hour, then take 1 mL of the lower liquid from the filter head and analyze it using a gas chromatograph.	+	[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.
−	<html>	+	<br>
−	<body>	+	[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.
−	<h3>Data Processing and Analysis</h3>	+	<br>
−	The experimental results indicated that the 4HB molar ratio achieved by introducing the pSEVA321 was higher than that of the pSEVA341. However, the dry weight decreased somewhat compared to the wild TD80.	+	[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.
	<br>		<br>
−	In order to stabilize the cell dry weight while increasing the 4HB molar ratio, we decided to explore the effect of the pSEVA321 on cell dry weight and make improvements. For further experiments, please turn to BBa_K5226075.	+	[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.
	<!-- Uncomment this to enable Functional Parameter display		<!-- Uncomment this to enable Functional Parameter display

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Latest revision as of 03:16, 1 October 2024

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.
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.