Difference between revisions of "Part:BBa K3606814"
 
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<h2>Design:</h2>
 
<h2>Design:</h2>
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Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we wanted to know whether one promoter can start three consecutive genes. We successfully cloned McbBCD into the pGEX plasmid together downstream one promoter and transcribed it into BL21. After 5 hours of induction of McbG expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbG product was produced.
+
Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we wanted to know whether one promoter can start three consecutive genes. We successfully cloned McbBCD into the pGEX plasmid together downstream one promoter and transcribed it into BL21. After 5 hours of induction of McbBCD expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbBCD product was produced.
  
 
<h2>Results:</h2>
 
<h2>Results:</h2>
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https://2020.igem.org/wiki/images/2/22/T--Fudan--img_McbA-E-G-BCD.jpeg
 
  
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Figure 1. SDS-PAGE result. lane 1: mcbA lane 2: mcbA+IPTG lane 3: mcbE lane 4: mcbE+IPTG  lane 5: mcbG  lane 6: mcbG+IPTG  lane 7: mcbBCD  lane 8: mcbBCD+IPTG lane 9: pGEX+ IPTG
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We have successfully expressed mcbC for the fundamental validation of the antimicrobial peptide(mccb17) expression.
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We've constructed the plasmid for mcbB and mcbD expression, yet the results of colony PCR is false positive thus further validation is needed.
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https://2020.igem.org/wiki/images/f/f3/T--Fudan--Poster_Mcb-gels.jpg
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Figure1. gel map for mcbA, mcbC, mcbE and mcbF
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Comparing the 3th and 4th lanes, after IPTG induction,the difference is small. This may be due to the low expression of McbBCD products. We still need to experiment more to find the problem.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:42, 5 December 2020


mcbBCD

Usage and Biology:

This part is an antibiotic coding gene cluster with proteins for MccB17 maturation.

Design:

Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we wanted to know whether one promoter can start three consecutive genes. We successfully cloned McbBCD into the pGEX plasmid together downstream one promoter and transcribed it into BL21. After 5 hours of induction of McbBCD expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbBCD product was produced.

Results:

We have successfully expressed mcbC for the fundamental validation of the antimicrobial peptide(mccb17) expression. We've constructed the plasmid for mcbB and mcbD expression, yet the results of colony PCR is false positive thus further validation is needed. T--Fudan--Poster_Mcb-gels.jpg Figure1. gel map for mcbA, mcbC, mcbE and mcbF


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2062
    Illegal PstI site found at 2095
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2062
    Illegal PstI site found at 2095
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1969
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2062
    Illegal PstI site found at 2095
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2062
    Illegal PstI site found at 2095
    Illegal NgoMIV site found at 1724
    Illegal AgeI site found at 1896
  • 1000
    COMPATIBLE WITH RFC[1000]