Difference between revisions of "Part:BBa K3331014"
 
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<div>[[File:T--XJTU-China--EPcircuit.png |600px|thumb|center|<b>Figure 2:</b>Designed verification circuit.]]</div>
 
<div>[[File:T--XJTU-China--EPcircuit.png |600px|thumb|center|<b>Figure 2:</b>Designed verification circuit.]]</div>
 
===Characterization===
 
===Characterization===
−
The test plasmid was constructed by Golden Gate Assembly and was confirmed by colony PCR. The length of B.S. galU fragment is 951bp, and the length of E.coli pgmA is 1770 bp. The following results showed successful constructions.
+
The test plasmid was constructed by Golden Gate Assembly and was confirmed by colony PCR. The length of the E.coli galU fragment is 981bp, and the length of P.A. pgmA is 1521bp. The following results showed successful constructions.
 
<div>[[File:T--XJTU-China--EPgel.png |700px|thumb|center|<b>Figure 1:</b>PCR confirmation of the designed plasmid.]]</div>
 
<div>[[File:T--XJTU-China--EPgel.png |700px|thumb|center|<b>Figure 1:</b>PCR confirmation of the designed plasmid.]]</div>
  
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<div>[[File:T--XJTU-China--EPrna.png |600px|thumb|center|<b>Figure 2:</b>RT-qPCR result of pgmA and galU.]]</div>
 
<div>[[File:T--XJTU-China--EPrna.png |600px|thumb|center|<b>Figure 2:</b>RT-qPCR result of pgmA and galU.]]</div>
  
−
Anthrone sulfuric acid method was used to detect EPS yields. Compared with the blank strain, the EPS yield of the engineering strain increased by 37.7%. 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.
+
Anthrone sulfuric acid method was used to detect EPS yields. Compared with the blank strain, the EPS yield of the engineering strain increased by 61.6%. The results of this study clearly show that it is possible to enhance the production of EPSs in DH5α by altering the expression of P.A. pgmA and E.coli galU.
  
 
<div>[[File:T--XJTU-China--EPEPS.png |600px|thumb|center|<b>Figure 3:</b>Detection of the expression of EPS.]]</div>
 
<div>[[File:T--XJTU-China--EPEPS.png |600px|thumb|center|<b>Figure 3:</b>Detection of the expression of EPS.]]</div>

Latest revision as of 21:28, 26 October 2020


E.coli galU+P.A 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. 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1587
    Illegal BglII site found at 1620
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 942
    Illegal AgeI site found at 1176
    Illegal AgeI site found at 1815
    Illegal AgeI site found at 2193
  • 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 the E.coli galU fragment is 981bp, and the length of P.A. pgmA is 1521bp. 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 61.6%. The results of this study clearly show that it is possible to enhance the production of EPSs in DH5α by altering the expression of P.A. pgmA and E.coli galU.

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.