Difference between revisions of "Part:BBa K2921400"
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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. | 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 | + | <b>Construct design</b> |
(insert photo) | (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. | 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 | + | <b>PCR</b> |
PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct. | PCR check results, using the primers VF2 and VR, and sequencing results by Tri-I Biotech confirm this construct. | ||
(insert photo) | (insert photo) | ||
| − | Characterization | + | <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 ___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. | ||
Revision as of 08:03, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]