Difference between revisions of "Part:BBa K1983006"
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<h3>Expression assays</h3> | <h3>Expression assays</h3> | ||
− | The gp45 mutant protein expression was tested in Escherichia coli BL21; ER2566 and C41 strains, with | + | The gp45 mutant protein expression was tested in Escherichia coli BL21; ER2566 and C41 strains, with BL21 (DE3) strain showing the best results (fig. 2). <br> |
− | [[File:T--Vilnius-Lithuania--gp45gel.png|thumbnail|center|500px| <b>Figure | + | [[File:T--Vilnius-Lithuania--gp45gel.png|thumbnail|center|500px| <b>Figure 2. 12% SDS-PAGE analysis of modified Gp45 protein expression levels. </b> E. coli BL21 (DE3) was induced by IPTG (2 µM final concentration) using pET expression system. Red star indicated the weight of target protein. Odd numbers represent soluble protein fraction, even numbers represent insoluble fraction.]]<br><br> |
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Revision as of 15:45, 20 October 2016
gp45 phenylalanine mutant M37 with C-terminal 6XHis-Tag
T4 phage clamp protein gp45 with M37 stable mutations to phenylalanine.
Overview
The gp45 phenyalanine mutants belong to PolyPhe protein family used to condense free phenylalanine from the. PolyPhe proteins are distinctive for their high rates of phenylalanine. This protein coding biobrick part among with other twins was created to assess the bacteria‘s capability to synthesize phenylalanine-rich proteins. There are five mutants of gp45 ranging from 6 to 37 mutations changing five canonical aromatic or hydrophobic amino acids (Tyr, Trp, Leu, Ile, Met) to phenylalanine (see design page for further information). The whole range spans from 4,8% in the wild type protein to 21,2% of phenylalanine in gp45m37 protein.
This version of mutant has 37 mutations to phenylalanine, resulting in a total of 21,2%.
Experiments and Results
Cloning
The received sequences were amplified using Uni-FW/RV primers and digested with NdeI and XhoI. The fragments containing mutant genes were cloned into pET21b expression vector digested with the same restriction enzymes. Transformant colonies were PCR-screened using Prom7/Term7 primers and positive clone plasmids were sequenced prior to further usage.
Expression assays
The gp45 mutant protein expression was tested in Escherichia coli BL21; ER2566 and C41 strains, with BL21 (DE3) strain showing the best results (fig. 2).
![](/wiki/images/3/3d/T--Vilnius-Lithuania--gp45gel.png)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 688
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]