Difference between revisions of "Part:BBa K2705007"
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| − | TetA is a tetracycline resistance protein[TetA(C) inner-membrane-associated protein]. Codon sequence was opimized for Bacillus amyloliquefaciens | + | TetA is a tetracycline resistance protein[TetA(C) inner-membrane-associated protein]. Codon sequence was opimized for <i>Bacillus amyloliquefaciens</i>,so that its expression can be enhanced and function better. |
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Here we entrust Genescript to help us do the optimization. In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in <i>Bacillus amyloliquefaciens</i> FZB42 by upgrading the CAI from 0.69 to 0.92. (Fig. 2) GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. (Fig. 3) The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. In addition, our optimization process has screened and successfully modified several negative cis-acting sites. | Here we entrust Genescript to help us do the optimization. In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in <i>Bacillus amyloliquefaciens</i> FZB42 by upgrading the CAI from 0.69 to 0.92. (Fig. 2) GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. (Fig. 3) The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. In addition, our optimization process has screened and successfully modified several negative cis-acting sites. | ||
| − | [[File: Codon usage bias adjustment.png|600px|center|thumb| | + | [[File: Codon usage bias adjustment.png|600px|center|thumb|]] |
| − | [[File: GC Content Adjustment.png|600px|center|thumb| | + | [[File: GC Content Adjustment.png|600px|center|thumb|]] |
Latest revision as of 17:48, 17 October 2018
TetA (optimized for Bacillus amyloliquefaciens)
TetA is a tetracycline resistance protein[TetA(C) inner-membrane-associated protein]. Codon sequence was opimized for Bacillus amyloliquefaciens,so that its expression can be enhanced and function better.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 684
Illegal NgoMIV site found at 951
Illegal NgoMIV site found at 1146
Illegal AgeI site found at 180
Illegal AgeI site found at 219
Illegal AgeI site found at 279
Illegal AgeI site found at 441 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 216
Usage and biology
The tetracyclines (Tcs) is a group of antibiotics that have been widely used since the 1940s against both Gram-negative and Gram-positive bacteria. Resistance to Tcs has many mechanisms including the active efflux system of tetracycline, target gene mutations, enzymatic degradation of antibiotics, decreased drug permeability and so on. The tetracycline efflux pumps are encoded by several genes, including tetA, tetC, tetE, tetG, and tetH, which have been reported a major mechanism of Tcs resistance.
Design
Here we improved tetA (BBa_J31006) by codon optimizing for Bacillus amyloliquefaciens, which could result in Tcs resistance by encoding membrane-associated proteins that pump Tcs out of the cell, reducing intracellular drug concentrations and protecting intracellular ribosomes. Here we entrust Genescript to help us do the optimization. In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in Bacillus amyloliquefaciens FZB42 by upgrading the CAI from 0.69 to 0.92. (Fig. 2) GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. (Fig. 3) The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken. In addition, our optimization process has screened and successfully modified several negative cis-acting sites.
