Difference between revisions of "Part:BBa K4182009"
 
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we selected the E.coli-pgmA+E.coli-GalU gene (later referred to as EE gene) with the best EPS expression  with the LacI manipulator  to form plasmid IV  where the GalU gene  and the pgmA 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  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 high copy pSEVA341 plasmid and re-linked it to obtain new plasmid IV and successfully constructed it
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Extracellular polysaccharide EPS can be used for water conservation and sand fixation of soil. We designed and constructed EPS synthesis circuit by overexpression of pgmA (phosphoglucose mutase)and galU (UDP glucose pyrophosphorylase) genes, and conducted some optimizations and improvements based on previous work by team 2020-XJTU-China
  
 
<!-- Add more about the biology of this part here
 
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===Usage&Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4182009 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4182009 parameters</partinfo>
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<!-- -->
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==Profile==
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===Base Pairs===
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6356
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==Usage&Biology==
 
==Usage&Biology==
  
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===The original components are replaced with carriers to achieve more efficient expression===
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Our EPS synthesis circuit was constructed based on the previous work by team 2020-XJTU-China (BBa_K3331012)by the promoter and replicon optimization for the enhanced EPS synthesis. It contains the key enzymes GalU and PmgA under the regulation of LacI-Ptrc promoter. The EPS synthesis circuit was as below:
  
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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.
 
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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.
 
  
 
[[File:XJTU-p2-figure1.png|400px]]
 
[[File:XJTU-p2-figure1.png|400px]]
  
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Figure 1
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Figure 1 EPS synthesis circuit
  
 
===1. Introduction to extracellular polysaccharide synthesis===
 
===1. Introduction to extracellular polysaccharide synthesis===
  
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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]
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Figure 2 shows the EPS biosynthetic pathway: Glucose can be converted to glucose-1-phosphate via EMP pathway or by enzyme phosphoglucose mutase (PgmA), and then UDP is synthesized by UDP glucose pyrophosphorylase (GalU) and served as precursor of EPS. Fredrik at al reported that the overexpression of pgmA and galU genes in Streptococcus thermophiles enhanced the EPS production from 0.17 g/mol to 0.31 g/mol. The same strategy was also employed in our study and the team 2020-XJTU-China. (https://2020.igem.org/Team:XJTU-China/Engineering)
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https://2020.igem.org/Team:XJTU-China/Engineering
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[[File:XJTU-p2-figure2.png|400px]]
 
[[File:XJTU-p2-figure2.png|400px]]
  
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Figure 2  
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Figure 2 EPS biosynthetic pathway
  
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===2. Construction and validation of extracellular polysaccharide synthesis plasmid 4===
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===2. Construction and validation of EPS synthesis plasmid 4===
  
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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]
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Based on the work of the XJTU-2020 team, we selected the pgmA from E.coli (GenBank: CP041425.1) and galU gene from E.coli (GenBank: CP104721.1) which proved highest EPS production in previous study. LacI-Plac promoter fragment, pgmA and galU amplified from previous EE plasmid, were ligated into the backbone pSB1K3 by Golden Gate Assembly (Figure 3). Several clones were obtained after assembly and transformation, yet it is difficult to extract recombinant plasmid after several attempts. So another high copy number vector backbone pSEVA341 was employed, and the recombinant plasmid successfully constructed and verified by colony PCR as shown in Figure 4, and further confirmed by sequencing. The iGEM ID for plasmid 4 is BBa_K4182009.
  
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[[File:XJTU-p2-figure3.png|400px]]
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[[File:XJTU-p2-figure6.png|350px]]
  
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Figure 3: 2022 experimental group plasmids       
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Figure 3: PCR fragments used for construction of plasmid 4
  
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[[File:XJTU-p2-figure4.png|400px]]
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[[File:XJTU-4-3.png|400px]]
  
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Figure 4: 2020 EPS expression molar concentration comparison
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Figure 4: Colony PCR verification of plasmid 4
  
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[[File:XJTU-p2-figure5.png|400px]]
 
  
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Figure 5 (colony PCR) modified plasmid IV was successfully gelled
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===3.Improvements compared to 2020-XJTU-China team===
  
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[[File:XJTU-p2-figure6.png|400px]]
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As shown in the Figure 5, compared to the EE plasmid constructed by team 2020-XJTU-China, our EPS synthesis plasmid were constructed with replicon optimization from medium-copy-number pMB1ori (15-20 copies) of EE plasmid to high-copy-number pRO1600 ori (100-150 copies) of pSEVA341 vector, and promoter optimization from low efficient P43 promoter to inducible high efficient PlacUV5. The optimization leads to the increased transcriptional levels for both galU and pgmA gene as shown in Figure 13. After IPTG induction, the expression levels of galU and pgmA gene were 3.4 and 2.8 fold compared to that of EE plasmid, demonstrating the improvement after promoter and replicon optimization.
  
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Figure 6: pgmA and GalU gene extraction gum map
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[[File:XJTU-p4-14.png|400px]]
  
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[[File:XJTU-p2-figure7.png|400px]]
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Figure 5: Optimization of plasmid 4 compared to EE plasmid
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(BBa_K3331012  Vs  BBa_K4182009)
  
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Figure 7: Lac gene gum map
 
  
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[[File:XJTU-p2-figure9.png|400px]]
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[[File:XJTU-P4-10.png|400px]]
  
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Figure 8: pSEVA341 plasmid map
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Figure 6 RT-qPCR result of plasmid 4 compared to previous EE plasmid
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(IPTG was induced at a concentration of 1mM for 6h at 37℃.)
  
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===3. Improvements to the 2020 iGEM team===
 
  
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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.
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===4.Determination of EPS content===
  
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[[File:XJTU-p2-figure9.png|400px]]
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Anthrone-sulfuric acid colorimetry is a fast and convenient sugar fixation method. Under strong acidic conditions, anthrone can be associated with free or polysaccharides present in hexose, pentose and hexoclonic acid to generate blue-green glycal derivatives, and sugar content can be determined by the different color of glycal product, whose maximum absorption peak at 620 nm. In this experiment we can quantitatively compare the EPS content of engineered bacteria by this method.
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First, we used glucose at concentrations of 0, 0.2, 0.4, 0.6, and 0.8 (units of mg/ml) as standard solutions to determine the standard curve at 620 nm. (Figures 7 and 8)
  
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Figure 9: Team 2020 Plasmids
 
  
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[[File:XJTU-P4-11.png|400px]] [[File:XJTU-P4-12.png|400px]]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4182009 SequenceAndFeatures</partinfo>
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Figure 7-8: Standard curve using glucose as a standard solution and their different colors.
  
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In order to avoid the influence of the sugar in LB medium, we purified the the intracellular EPS by alcohol precipitation and re-dissolved in water.  The EPS content was detected by anthrone-sulfuric acid method and the results are shown in Figure 9.
  
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
+
[[File:XJTU-P4-13.png|400px]]
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<partinfo>BBa_K4182009 parameters</partinfo>
+
 
−
<!-- -->
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Figure 9: 620 nm detection S of three strains harboring EE plasmid or plasmid 4. (calculated as the ratio of absorbance to liquid concentration), *indicating that there are significant differences in this sample compared to EE (based on T-TEST).
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Compared with the previous EE plasmid, the EPS yield of the engineering bacteria with plasmid 4 has been significantly improved by about 3 fold (P<0.05), demonstrating the construction of plasmid 4 is successful and effective, and also confirming our improvement.
 +
 
 +
 
 +
==References==
 +
 
 +
[1]Rafael Silva-Rocha, Esteban Martínez-García, Belén Calles, Max Chavarría, Alejandro Arce-Rodríguez, Aitor de las Heras, A. David Páez-Espino, Gonzalo Durante-Rodríguez, Juhyun Kim, Pablo I. Nikel, Raúl Platero, Víctor de Lorenzo, The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes, Nucleic Acids Research, Volume 41, Issue D1, 1 January 2013, Pages D666–D675, https://doi.org/10.1093/nar/gks1119
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 +
[2]Jorge Alonso-Gutierrez, Rossana Chan, Tanveer S. Batth, Paul D. Adams, Jay D. Keasling, Christopher J. Petzold, Taek Soon Lee, Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production, Metabolic Engineering, Volume 19,2013, Pages 33-41, ISSN 1096-7176,https://doi.org/10.1016/j.ymben.2013.05.004.
 +
 
 +
[3]Figurski, D.H. and Helinski, D.R. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl Acad. Sci. USA, 76, 1648–1652.
 +
 
 +
[4]Santos,P.M., Di Bartolo,I., Blatny,J.M., Zennaro,E. and Valla,S.(2001) New broad-host
 +
-range promoter probe vectors based on the plasmid RK2 replicon. FEMS Microbiol. Lett., 195, 91–96.
 +
 
 +
[5]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.

Latest revision as of 04:10, 14 October 2022


A circuit for efficient exopolysaccharide synthesis


Extracellular polysaccharide EPS can be used for water conservation and sand fixation of soil. We designed and constructed EPS synthesis circuit by overexpression of pgmA (phosphoglucose mutase)and galU (UDP glucose pyrophosphorylase) genes, and conducted some optimizations and improvements based on previous work by team 2020-XJTU-China

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4724
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4724
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4724
    Illegal BglII site found at 4733
    Illegal BglII site found at 7225
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4724
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Base Pairs

6356

Usage&Biology

Our EPS synthesis circuit was constructed based on the previous work by team 2020-XJTU-China (BBa_K3331012)by the promoter and replicon optimization for the enhanced EPS synthesis. It contains the key enzymes GalU and PmgA under the regulation of LacI-Ptrc promoter. The EPS synthesis circuit was as below:


XJTU-p2-figure1.png

Figure 1 EPS synthesis circuit

1. Introduction to extracellular polysaccharide synthesis

Figure 2 shows the EPS biosynthetic pathway: Glucose can be converted to glucose-1-phosphate via EMP pathway or by enzyme phosphoglucose mutase (PgmA), and then UDP is synthesized by UDP glucose pyrophosphorylase (GalU) and served as precursor of EPS. Fredrik at al reported that the overexpression of pgmA and galU genes in Streptococcus thermophiles enhanced the EPS production from 0.17 g/mol to 0.31 g/mol. The same strategy was also employed in our study and the team 2020-XJTU-China. (https://2020.igem.org/Team:XJTU-China/Engineering)

XJTU-p2-figure2.png

Figure 2 EPS biosynthetic pathway

2. Construction and validation of EPS synthesis plasmid 4

Based on the work of the XJTU-2020 team, we selected the pgmA from E.coli (GenBank: CP041425.1) and galU gene from E.coli (GenBank: CP104721.1) which proved highest EPS production in previous study. LacI-Plac promoter fragment, pgmA and galU amplified from previous EE plasmid, were ligated into the backbone pSB1K3 by Golden Gate Assembly (Figure 3). Several clones were obtained after assembly and transformation, yet it is difficult to extract recombinant plasmid after several attempts. So another high copy number vector backbone pSEVA341 was employed, and the recombinant plasmid successfully constructed and verified by colony PCR as shown in Figure 4, and further confirmed by sequencing. The iGEM ID for plasmid 4 is BBa_K4182009.

XJTU-p2-figure6.png

Figure 3: PCR fragments used for construction of plasmid 4

XJTU-4-3.png

Figure 4: Colony PCR verification of plasmid 4


3.Improvements compared to 2020-XJTU-China team

As shown in the Figure 5, compared to the EE plasmid constructed by team 2020-XJTU-China, our EPS synthesis plasmid were constructed with replicon optimization from medium-copy-number pMB1ori (15-20 copies) of EE plasmid to high-copy-number pRO1600 ori (100-150 copies) of pSEVA341 vector, and promoter optimization from low efficient P43 promoter to inducible high efficient PlacUV5. The optimization leads to the increased transcriptional levels for both galU and pgmA gene as shown in Figure 13. After IPTG induction, the expression levels of galU and pgmA gene were 3.4 and 2.8 fold compared to that of EE plasmid, demonstrating the improvement after promoter and replicon optimization.

XJTU-p4-14.png

Figure 5: Optimization of plasmid 4 compared to EE plasmid (BBa_K3331012 Vs BBa_K4182009)


XJTU-P4-10.png

Figure 6 RT-qPCR result of plasmid 4 compared to previous EE plasmid (IPTG was induced at a concentration of 1mM for 6h at 37℃.)


4.Determination of EPS content

Anthrone-sulfuric acid colorimetry is a fast and convenient sugar fixation method. Under strong acidic conditions, anthrone can be associated with free or polysaccharides present in hexose, pentose and hexoclonic acid to generate blue-green glycal derivatives, and sugar content can be determined by the different color of glycal product, whose maximum absorption peak at 620 nm. In this experiment we can quantitatively compare the EPS content of engineered bacteria by this method. First, we used glucose at concentrations of 0, 0.2, 0.4, 0.6, and 0.8 (units of mg/ml) as standard solutions to determine the standard curve at 620 nm. (Figures 7 and 8)


XJTU-P4-11.png XJTU-P4-12.png

Figure 7-8: Standard curve using glucose as a standard solution and their different colors.

In order to avoid the influence of the sugar in LB medium, we purified the the intracellular EPS by alcohol precipitation and re-dissolved in water. The EPS content was detected by anthrone-sulfuric acid method and the results are shown in Figure 9.


XJTU-P4-13.png

Figure 9: 620 nm detection S of three strains harboring EE plasmid or plasmid 4. (calculated as the ratio of absorbance to liquid concentration), *indicating that there are significant differences in this sample compared to EE (based on T-TEST).

Compared with the previous EE plasmid, the EPS yield of the engineering bacteria with plasmid 4 has been significantly improved by about 3 fold (P<0.05), demonstrating the construction of plasmid 4 is successful and effective, and also confirming our improvement.


References

[1]Rafael Silva-Rocha, Esteban Martínez-García, Belén Calles, Max Chavarría, Alejandro Arce-Rodríguez, Aitor de las Heras, A. David Páez-Espino, Gonzalo Durante-Rodríguez, Juhyun Kim, Pablo I. Nikel, Raúl Platero, Víctor de Lorenzo, The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes, Nucleic Acids Research, Volume 41, Issue D1, 1 January 2013, Pages D666–D675, https://doi.org/10.1093/nar/gks1119

[2]Jorge Alonso-Gutierrez, Rossana Chan, Tanveer S. Batth, Paul D. Adams, Jay D. Keasling, Christopher J. Petzold, Taek Soon Lee, Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production, Metabolic Engineering, Volume 19,2013, Pages 33-41, ISSN 1096-7176,https://doi.org/10.1016/j.ymben.2013.05.004.

[3]Figurski, D.H. and Helinski, D.R. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl Acad. Sci. USA, 76, 1648–1652.

[4]Santos,P.M., Di Bartolo,I., Blatny,J.M., Zennaro,E. and Valla,S.(2001) New broad-host -range promoter probe vectors based on the plasmid RK2 replicon. FEMS Microbiol. Lett., 195, 91–96.

[5]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.