Part:BBa_K4182009:Design
A circuit for efficient exopolysaccharide synthesis
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4724
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4724
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4724
Illegal BglII site found at 4733
Illegal BglII site found at 7225 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4724
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4724
Illegal NgoMIV site found at 1103
Illegal NgoMIV site found at 4610
Illegal AgeI site found at 6785
Illegal AgeI site found at 6951 - 1000COMPATIBLE WITH RFC[1000]
Profile
Base Pairs
6356
Design Notes
we found that using the pSEVA341 plasmid Replacing the medium-high copy pSB1K3 plasmid as a vector resulted in a higher degree of expression as well as a more significant stability of gene expression The pSEVA plasmid allows direct binding to express heterologous genes and retains polyclonal sites after binding to any gene. This modularity and compatibility with various replicons allows the assembly of complex circuits in the same host, and the ease of monitoring and modular control of each subcircuit helps ease the transition from trial-and-error genetic engineering to systematic synthetic biology. It is more beneficial for the characterization and practical application of modular research in synthetic biology. [2]We also designed the introduction of LacI regulatory protein to obtain the target gene expression product more accurately and efficiently, and to achieve better ability to regulate the product to achieve water fixation and moisture retention.
Usage&Biology
The original components are replaced with carriers to achieve more efficient expression
To facilitate the modularized design of plasmids, we named the EPS synthesis verification plasmid 4, which will be referred to as plasmid 4 in the following paragraphs. Plasmid 4 is a gene vector for the synthesis of EPS extracellular polysaccharides. The main gene progenitor contains the EPS synthesis gene, LacI regulatory protein synthesis gene + Ptrc promoter, as shown in Figure 1.
Figure 1
1. Introduction to extracellular polysaccharide synthesis
We hope to produce extracellular polysaccharide EPS in engineered bacteria by plasmid IV to achieve soil fixation and moisture retention, and the principle of EPS synthesis by plasmid IV in bacteria is that: glucose enters the hexose diphosphate pathway (EMP) or synthesizes glucose-1-phosphate catalyzed by PGM, and then UDP is synthesized by UDP glucose pyrophosphorylase (galU). UDP glucose can be used as a raw material for synthesizing EPS. Also after reviewing the literature, we found that Fredrik Levander and his team overexpressed pgmA and galU genes in Streptococcus thermophilus[1]. They found that EPS production in Streptococcus thermophilus increased from 0.17 g/mol to 0.31 g/mol when galU and pgmA (encoding phosphoglucose mutase (PGM)) were overexpressed, and we hypothesized that overexpression of pgmA and galU genes in bacteria would also increase extracellular polysaccharide production. Therefore, we wanted to overexpress pgmA gene and galU gene in engineered bacteria to achieve high extracellular polysaccharide production[1], the principle of which is shown in Figure 2.[1] https://2020.igem.org/Team:XJTU-China/Engineering
Figure 2
2. Construction and validation of extracellular polysaccharide synthesis plasmid 4
Based on the work of the XJTU-2020 team, we selected the E.coli-pgmA+E.coli-Galu gene (later referred to as EE gene) with the best EPS expression (as shown in Figure IV ) with the LacI manipulator (GenBank: NC_000913.3 ) to form plasmid IV (Figure III ) where the GalU gene (sequence GenBank: CP104721.1) and the pgmA（GenBank: CP041425.1） gene set from E. coli were synthesized into the EE gene, In specific experiments, we obtained the EE gene, LacI gene in separate isolation and extraction (Figure In our specific experiments, we isolated and extracted EE gene, LacI gene (Figure 6 and 7) pSB1K3 plasmid vector and recovered them, and then performed GoldenGate ligation by using Bsa I enzyme cleavage site. Because the plasmid copy number was not high, we were unable to obtain the linker. So we changed to a high copy pSEVA341 plasmid and re-linked it to obtain new plasmid IV and successfully constructed it (Figure iii). pSEVA plasmid allows direct binding to express heterologous genes and retains polyclonal sites after binding to any gene. This modularity and compatibility with various replicons allow the assembly of complex circuits in the same host, and the ease of monitoring and modular control of each subcircuit helps ease the transition from trial-and-error genetic engineering to systematic synthetic biology. It is more beneficial for the characterization and practical application of modular research in synthetic biology.[2]
Figure 3: 2022 experimental group plasmids
Figure 4: 2020 EPS expression molar concentration comparison
Figure 5 (colony PCR) modified plasmid IV was successfully gelled
Figure 6: pgmA and GalU gene extraction gum map
Figure 7: Lac gene gum map
Figure 8: pSEVA341 plasmid map
3. Improvements to the 2020 iGEM team
Compared to the plasmids designed by the 2020 team (e.g., Figure IX), we found that using the pSEVA341 plasmid (e.g., Figure VIII) Replacing the medium-high copy pSB1K3 plasmid as a vector resulted in a higher degree of expression as well as more significant stability of gene expression (as in Figure IV ) The pSEVA plasmid allows direct binding to express heterologous genes and retains polyclonal sites after binding to any gene. This modularity and compatibility with various replicons allow the assembly of complex circuits in the same host, and the ease of monitoring and modular control of each subcircuit helps ease the transition from trial-and-error genetic engineering to systematic synthetic biology. It is more beneficial for the characterization and practical application of modular research in synthetic biology. [2]We also designed the introduction of LacI regulatory protein to obtain the target gene expression product more accurately and efficiently and to achieve a better ability to regulate the product to achieve water fixation and moisture retention.
RT-qPCR comparison chart
EPS production comparison chart
Figure 9: Team 2020 Plasmids
References
[1] LEVANDER F, SVENSSON M, RåDSTRöM P. Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus [J]. Applied and Environmental Microbiology, 2002, 68(2): 784-90.
[2] SILVA-ROCHA R, MARTINEZ-GARCIA E, CALLES B, et al. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes [J]. Nucleic Acids Res, 2013, 41(Database issue): D666-75.