Difference between revisions of "Part:BBa K4182009"
 
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Figure 2 EPS biosynthetic pathway
 
Figure 2 EPS biosynthetic pathway
  
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===2. Construction and validation of EPS 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 (https://2020.igem.org/Team:XJTU-China/Engineering), 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 10). 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 11, and further confirmed by sequencing. The iGEM ID for plasmid 4 is BBa_K4182009.
+
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-figure6.png|350px]]
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[[File:XJTU-p2-figure6.png|400px]]
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Figure 3: PCR fragments used for construction of plasmid 4
 
Figure 3: PCR fragments used for construction of plasmid 4
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Figure 4: Colony PCR verification of plasmid 4
 
Figure 4: Colony PCR verification of plasmid 4
  
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 +
===3.Improvements compared to 2020-XJTU-China team===
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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.
  
 
[[File:XJTU-p4-14.png|400px]]
 
[[File:XJTU-p4-14.png|400px]]
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Figure 5: Optimization of plasmid 4 compared to EE plasmid
 
Figure 5: Optimization of plasmid 4 compared to EE plasmid
 
(BBa_K3331012  Vs  BBa_K4182009)
 
(BBa_K3331012  Vs  BBa_K4182009)
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−
 
−
===3.Improvements compared to 2020-XJTU-China team===
 
−
 
−
As shown in the FIG12, 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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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.
 
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. (FIG 14)
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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)
  
  
 
[[File:XJTU-P4-11.png|400px]] [[File:XJTU-P4-12.png|400px]]
 
[[File:XJTU-P4-11.png|400px]] [[File:XJTU-P4-12.png|400px]]
 
 
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FIG 7-8: Standard curve using glucose as a standard solution and their different colors.
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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 FIG8.
+
  
 +
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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[[File:XJTU-P4-13.png|400px]]
 
  
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FIG 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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[[File:XJTU-P4-13.png|400px]]
  
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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).
  
 
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.
 
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.

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.