Difference between revisions of "Part:BBa K2921400"

 
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<partinfo>BBa_K2921400 short</partinfo>
 
<partinfo>BBa_K2921400 short</partinfo>
  
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This construct constitutively expresses MBP.  
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This construct constitutively expresses a colored metal-binding protein MBP (Basic part: BBa_K346004) MBP is a lead binding peptide that can accumulate lead ions from aquatic environment.
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 +
Construct design
 +
(insert photo)
 +
We inserted an upstream constitutive strong promoter-RBS combination (Basic part: BBa_K880005) and a downstream strong double terminator (Basic part: BBa_B0015) so as to maximize plasmid and protein expression.
 +
 
 +
PCR
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 +
PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct.
 +
(insert photo)
 +
 
 +
Characterization
 +
 
 +
To verify MBP expression in E. coli, we ran a SDS-PAGE gel, expecting a protein band at ___kDa. We prepared and lysed overnight liquid cultures of cells expressing MBP-mRFP, MBP only, and mRFP only. The SDS-PAGE reveals a darker band at ___ kDa as compared to the MBP-only and mRFP-only controls.
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(insert photo)
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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 07:54, 18 October 2019


Strong Promoter + RBS + MBP + Double Terminator

This construct constitutively expresses a colored metal-binding protein MBP (Basic part: BBa_K346004) MBP is a lead binding peptide that can accumulate lead ions from aquatic environment.

Construct design (insert photo) We inserted an upstream constitutive strong promoter-RBS combination (Basic part: BBa_K880005) and a downstream strong double terminator (Basic part: BBa_B0015) so as to maximize plasmid and protein expression.

PCR

PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct. (insert photo)

Characterization

To verify MBP expression in E. coli, we ran a SDS-PAGE gel, expecting a protein band at ___kDa. We prepared and lysed overnight liquid cultures of cells expressing MBP-mRFP, MBP only, and mRFP only. The SDS-PAGE reveals a darker band at ___ kDa as compared to the MBP-only and mRFP-only controls. (insert photo)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]