Part:BBa_K1506002

Optimized Superfolder GFP - only fast codons

Superfolder GFP has been modified so that the coding sequence contains all "fast" codons. The reason that we designed this version of superfolder GFP was to explore the possibilities with codon optimization. Currently, codon optimization focuses on whether the relative abundance of a codon in the genome. We have decided to extend this to whether codons for the specific amino acid are considered to have a fast translation rate or a slow translation rate; this variation of superfolder GFP has the fast coding sequence. We have modified the superfolder GFP so that there can be more expression of superfolder GFP.

Superfolder GFP (part BBa_I746916) has been modified so that the coding sequence contains only "fast" codons. This GFP was designed as one of five variants we optimized at the codon level using different criteria in order to develop a novel method for codon optimization. Traditional methods of codon optimization rely primarily on the distinction between degenerate codons that are common throughout the entire genome of E. coli and those that are rare. This GFP was optimized instead using only degenerate codons that are more frequent in higher translation initiation regions of the genome, ie fast codons. This criterion is based on the results of a recent project in which all the coding DNA sequences of E. coli are divided into five groups based on the naturally occurring TIR, from lowest to highest. Then, the codon usage profile of each group of genes is statistically analyzed to determine whether a codon is slow or fast. A fast codon is defined as one with high correlation between TIR and its frequency. Otherwise, it is a slow codon. Ng, C. Y., Farasat, I., Zomorrodi, A. R., Maranas, C. D. & Salis, H. M. Model-guided construction and optimization of synthetic metabolism for chemical product synthesis. Synthetic Biology Engineering Research Center Spring Retreat (2013), Berkeley, CA. It is hypothesized that the groups of CDS with high TIR will hold more “fast” codons, which will lead to higher translation elongation rate and thus higher protein expression, whereas the slow regions will hold more “slow” codons leading to lower expression. Characterization of this synthetic reporter gene showed robust expression across a wide range of translation initiation:

 
 This graph shows the absorbance of cells expressing fast superfolder GFP verses time.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 664
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 664
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 664
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 664
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 664
  
• 1000
  COMPATIBLE WITH RFC[1000]