Difference between revisions of "Part:BBa K2921400"

Revision as of 08:19, 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 17.48 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 17.48 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]

@@ Line 19: / Line 19: @@
 <b>Characterization</b>
-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.
+To verify MBP expression in E. coli, we ran a SDS-PAGE gel, expecting a protein band at 17.48 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 17.48 kDa as compared to the MBP-only and mRFP-only controls.
 (insert photo)