Difference between revisions of "Part:BBa K3606814"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3606814 short</partinfo>
 
<partinfo>BBa_K3606814 short</partinfo>
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<h2>Usage and Biology:</h2>
 
This part is an antibiotic coding gene cluster with  proteins for MccB17 maturation.  
 
This part is an antibiotic coding gene cluster with  proteins for MccB17 maturation.  
  
 
<h2>Design:</h2>
 
<h2>Design:</h2>
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Since we encountered difficulties in the expression of McbABCDEFG at the beginning, we decided to check whether the McbA/B/C/D/E/F/G genes can be expressed and translated separately. We successfully cloned McbG into the pGEX plasmid 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.
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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.
  
 
<h2>Results:</h2>
 
<h2>Results:</h2>
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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
 
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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Comparing the 5th and 6th lanes, after IPTG induction, there is a clear band, which is the product of McbG expression.
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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.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 01:45, 28 October 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 McbG expression with IPTG, the whole protein SDS-PAGE was used to detect whether the McbG product was produced.

Results:

T--Fudan--img_McbA-E-G-BCD.jpeg

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

Comparing the 3th and 4th lanes, after IPTG induction,the difference is small. This may be due to the low expression of McbBCD products.

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]