Part:BBa_K2705007

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  INCOMPATIBLE 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.