Difference between revisions of "Part:BBa K3331012"

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<html><img style="float:left;width:64px;margin-right:2em" src="https://static.igem.wiki/teams/4765/wiki/2023-b-home.png" alt="contributed by Fudan iGEM 2023"></html>
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===Improved by Fudan iGEM 2023 ===
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This composite part can increase the production of EPS in ''E coli''. Our team adopted this method for EPS production and verified its adhesion capability.
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Unlike a simple arrangement of CDSs, we have improved a [https://2023.igem.wiki/fudan/software/ ribozyme-assisted polycistronic co-expression system] by inserting stem-loop and Twister P1 sequences between CDSs.Through the Twister P1 ribozyme, the polycistronic mRNA containing galU and pgmA is cleaved into monocistronic mRNAs, each containing either galU or pgmA. The stem-loop prevents degradation of the mRNA after cleavage.
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We also added red fluorescent protein mScarlet at the third position of pRAP. We found EPS expressing bacteria is "heavier", precipitate faster, likely due to more "sticky". Before forcely pepitting, these EPS expressing bacteria form cluster in liquid culture. Under a fluorescence microscope, by increasing the flow speed of culture media, we observed that ''E. coli'' with red fluorescence (simultaneously expressing ''galU'' and ''pgmA'') were the very last ones being washed away, confirming superior adhesion capability.
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====Improved part====
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Our improved part is [https://parts.igem.org/Part:BBa_K4765121 BBa_K4765121 (ribozyme connected: galU + pgmA + mScarlet)].
  
 
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6356
 
6356
  
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===Design Notes===
 
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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==
 
==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-figure5.png|400px]]
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[[File:XJTU-4-3.png|400px]]
  
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Figure 4: (colony PCR) modified plasmid IV was successfully gelled
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Figure 4: Colony PCR verification of plasmid 4
  
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[[File:XJTU-p2-figure6.png|400px]]
 
  
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Figure 5: pgmA and GalU gene extraction gum map
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===3.Improvements compared to 2020-XJTU-China team===
  
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[[File:XJTU-p2-figure7.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: Lac gene gum map
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[[File:XJTU-p4-14.png|400px]]
 
[[File:XJTU-p4-14.png|400px]]
  
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Figure 7: Design of plasmid 4
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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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===3.Improvements to the 2020 iGEM team===
 
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As shown in the FIG6, compared to the EE plasmid constructed by team 2020-XJTU-China, our EPS synthesis plasmid showed increased transcriptional levels for both galU and pgmA gene. After IPTG induction, the expression levels of galU and pgmA gene were 3.4 and 2.8 fold compared to that of EE, demonstrating the improvement after promoter and replicon optimization.PSEVA plasmid allow direct expression of heterologous genes, still more after combined with any genetic cloning sites, this modular and various replicon compatibility allows assembly in the same host complex circuit, and each child circuit are easy to monitor and modular control help from trial-and-error type of genetic engineering to the system of synthetic biology. It is beneficial to the characteristic display and practical application of synthetic biology modular research.
 
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Comparison of RT-qPCR was shown below：
 
  
 
[[File:XJTU-P4-10.png|400px]]
 
[[File:XJTU-P4-10.png|400px]]
  
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IPTG was induced at a concentration of 1mM for 6h at 37℃.
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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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===4.Determination of EPS content===
 
===4.Determination of EPS content===
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Anthraxone colorimetry is a fast and convenient sugar fixation method, under strong acidic conditions, anthraconone can be associated with free or polysaccharides present in hexose, pentose and hexoclonic acid to generate blue-green glycal derivatives, the depth of its color and the sugar content in a certain range is proportional, the maximum absorption peak at 620nm. In this experiment we can semi-quantitatively compare the synthetic amount of EPS 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 7)
 
  
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[[File:XJTU-P4-11.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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[[File:XJTU-P4-12.png|400px]]
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[[File:XJTU-P4-11.png|400px]] [[File:XJTU-P4-12.png|400px]]
 
 
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FIG 8-9: Shows the color change of the standard curve data using glucose as a standard solution versus the different concentrations of the standard solution.
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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 exclude the influence of the sugars contained in LB medium on the experimental results during the experiment, we isolated the relatively pure bacteria by alcoholic deposition and detected the relative expression of the sugar content, and we showed the experimental results in FIG8.
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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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[[File:XJTU-P4-13.png|400px]]
 
[[File:XJTU-P4-13.png|400px]]
  
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FIG 10: Shows the relative absorbance of three different bacterial purification solutions at 620 nm (calculated as the ratio of absorbance to liquid concentration), *indicating that there are significant differences in this sample compared to EE bacteria (based on T-TEST).
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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 engineering bacteria introduced into the EE plasmid, the expression of polysaccharides introduced into the engineering bacteria of plasmid 4 has been significantly improved (P<0.05) We can consider that the part of the increase is due to the introduction of plasmid 4, the synthesis of additional EPS. From this we preliminarily prove that the construction of plasmid 4 is successful and effective.
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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==
 
==References==

Latest revision as of 11:04, 12 October 2023

contributed by Fudan iGEM 2023

Improved by Fudan iGEM 2023

This composite part can increase the production of EPS in E coli. Our team adopted this method for EPS production and verified its adhesion capability.

Unlike a simple arrangement of CDSs, we have improved a ribozyme-assisted polycistronic co-expression system by inserting stem-loop and Twister P1 sequences between CDSs.Through the Twister P1 ribozyme, the polycistronic mRNA containing galU and pgmA is cleaved into monocistronic mRNAs, each containing either galU or pgmA. The stem-loop prevents degradation of the mRNA after cleavage.

We also added red fluorescent protein mScarlet at the third position of pRAP. We found EPS expressing bacteria is "heavier", precipitate faster, likely due to more "sticky". Before forcely pepitting, these EPS expressing bacteria form cluster in liquid culture. Under a fluorescence microscope, by increasing the flow speed of culture media, we observed that E. coli with red fluorescence (simultaneously expressing galU and pgmA) were the very last ones being washed away, confirming superior adhesion capability.

Improved part

Our improved part is BBa_K4765121 (ribozyme connected: galU + pgmA + mScarlet).


E.coli galU+E.coli pgmA

This part can increase the yield of EPS. The yield of EPS can be significantly increased by overexpressing two key enzymes galU and pgmA at the same time. The part galU is from Escherichia coli YJ4. The part pgmA is from Escherichia coli str. K-12 substr. MG1655 Reference: Levander Fredrik et al. Applied and environmental microbiology,2002,68(2).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Overview

In order to increase the production of exopolysaccharides (EPSs), we altered the levels of enzymes in the central metabolism. We selected and overexpressed two key enzymes, galU and pgmA at the same time. And we found we successfully enhance the production of EPSs in E.coli.DH5α

Figure 1:Plasmid we have constructed for galU&pgmA validations.
Figure 2:Designed verification circuit.

Characterization

The test plasmid was constructed by Golden Gate Assembly and was confirmed by colony PCR. The length of E.coli galU fragment is 981bp, and the length of E.coli pgmA is 1770bp. The following results showed successful constructions.

Figure 1:PCR confirmation of the designed plasmid.

Our strain was cultured in ampicillin-resistant LB medium with strictly control variables. We stop the culture after 24 hours of culture. We measure the expression of EPS and the level of gene transcription during this period. RT-qPCR was conducted to test their expressions. And we can see these two genes were transcribed successfully.

Figure 2:RT-qPCR result of pgmA and galU.

Anthrone sulfuric acid method was used to detect EPS yields. Compared with the blank strain, the EPS yield of the engineering strain increased by 106.4%. The results of this study clearly show that it is possible to enhance the production of EPSs in DH5α by altering the expression of this part.

Figure 3:Detection of the expression of EPS.

We also measured the growth of DH5α transferred into the plasmid by enzyme labeling instrument. We measure the OD value of bacteria every hour under the wavelength of 600nm and fit the results. It was compared with the blankDH5α and the growth curve was drawn.

Figure 4:Growth curve of strains.

Improvement from XJTU-China-2022 iGEM

Profile

Name

A circuit for efficient exopolysaccharide synthesis

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.