Difference between revisions of "Part:BBa K1983007"
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<h2>Overview</h2> | <h2>Overview</h2> | ||
− | The Csm4 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 three mutants of Csm4 ranging from 11 to 42 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 7,7% in the first mutant to 21% of phenylalanine in the third mutant. | + | The Csm4 phenyalanine mutants belong to PolyPhe protein family used to condense free phenylalanine from the outer medium. 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 three mutants of Csm4 ranging from 11 to 42 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 7,7% in the first mutant to 21% of phenylalanine in the third mutant (Table 1.). |
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+ | [[File:T--Vilnius-Lithuania--csmlentele.png|thumbnail|center|500px| <b> Table 1.</b>: Comparison of phenylalanine quantities between the wild type and the mutant Csm4 proteins.]]<br><br> | ||
This version of mutant has 11 mutations to phenylalanine, resulting in a total of 7,7%. | This version of mutant has 11 mutations to phenylalanine, resulting in a total of 7,7%. | ||
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Synthetic PolyPhe proteins are enriched in phenylalanine and, therefore, a cell has to use more of this amino acid to produce the protein. As a result, cell’s pool of phenylalanine rapidly decreases. Consequently, a target amino acid from extracellular environment is absorbed in order to increase inner phenylalanine pool and continue translation of PolyPhe protein. As a result, cells demonstrate higher consumption of phenylalanine and its concentration outer environment decreases faster than normally. | Synthetic PolyPhe proteins are enriched in phenylalanine and, therefore, a cell has to use more of this amino acid to produce the protein. As a result, cell’s pool of phenylalanine rapidly decreases. Consequently, a target amino acid from extracellular environment is absorbed in order to increase inner phenylalanine pool and continue translation of PolyPhe protein. As a result, cells demonstrate higher consumption of phenylalanine and its concentration outer environment decreases faster than normally. | ||
− | We have characterized | + | We have characterized three protein mutants of the family (Csm4M11, Csm4M42 and Gp45M37) by evaluating the uptake of phenylalanine from the special substrate medium (fig. 3). |
[[File:T--Vilnius-Lithuania--gp45csm4invivo.png|thumbnail|center|500px| <b> Figure 3. Cell’s activity in reduction of 1.1 g of phenylalanine in growth medium (pH 7.4, 37ºC) while expressing PolyPhe over 30-minute period.</b> Control is cells without any biobrick. Gp45 (37) was expressed in E. coli BL21, Csm4 (11) and Csm4 (42) in E. coli ER2566. Results are achieved by 2.5 grams of cells.]]<br><br> | [[File:T--Vilnius-Lithuania--gp45csm4invivo.png|thumbnail|center|500px| <b> Figure 3. Cell’s activity in reduction of 1.1 g of phenylalanine in growth medium (pH 7.4, 37ºC) while expressing PolyPhe over 30-minute period.</b> Control is cells without any biobrick. Gp45 (37) was expressed in E. coli BL21, Csm4 (11) and Csm4 (42) in E. coli ER2566. Results are achieved by 2.5 grams of cells.]]<br><br> | ||
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Latest revision as of 09:22, 21 October 2016
Streptococcus thermofilus Csm4 phenylalanine mutant M11 with C-terminal 6XHis-Tag
Csm4 phenylalanine mutant M11 with C-terminal 6XHis-Tag
Overview
The Csm4 phenyalanine mutants belong to PolyPhe protein family used to condense free phenylalanine from the outer medium. 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 three mutants of Csm4 ranging from 11 to 42 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 7,7% in the first mutant to 21% of phenylalanine in the third mutant (Table 1.).
This version of mutant has 11 mutations to phenylalanine, resulting in a total of 7,7%.
Experiments and Results
Cloning
The received sequences were amplified using Uni-FW/RV primers and digested with Esp3I and XhoI. The fragments containing mutant genes were cloned into pETDuet™ expression vector digested with NcoI and XhoI. Transformant colonies were PCR-screened using Up-1A/Down-3 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 TOP10 strains, with ER2566 strain showing the best results (fig. 2).
![](/wiki/images/7/74/T--Vilnius-Lithuania--Csm4gel.png)
Characterization in vivo
Synthetic PolyPhe proteins are enriched in phenylalanine and, therefore, a cell has to use more of this amino acid to produce the protein. As a result, cell’s pool of phenylalanine rapidly decreases. Consequently, a target amino acid from extracellular environment is absorbed in order to increase inner phenylalanine pool and continue translation of PolyPhe protein. As a result, cells demonstrate higher consumption of phenylalanine and its concentration outer environment decreases faster than normally. We have characterized three protein mutants of the family (Csm4M11, Csm4M42 and Gp45M37) by evaluating the uptake of phenylalanine from the special substrate medium (fig. 3).
![](/wiki/images/d/d5/T--Vilnius-Lithuania--gp45csm4invivo.png)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 796
Illegal XhoI site found at 907 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]