Part:BBa_K1499203

SdaB protein generator without tRNA + 5 amber stops

This part generates the serine deaminase, SdaB (BBa_K847080), which confers basicity resistance with strong promoter/RBS pair (BBa_BBa_J23038), terminator (BBa_B0010). However, this part will not work without the supP tRNA submitted by Stanford-Brown-Spelman 2014 (BBa_K1499251).

Usage and Biology

Serine catabolism creates buffers in the cytoplasm to help counter the effects of the high pH. This enzyme will break down serine to confer resistance to higher pH. Please refer to Stanford-Brown 2012 iGEM's page on this enzyme's part, BBa_K847080.

This part is to be used in the Stanford-Brown-Spelman 2014 iGEM amberless system with the supP tRNA to confer resistance to higher pH. The stop codons inhibit the expression of the complete product, except in amberless cells, that do not have the UAG release factors.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 6
  Illegal NheI site found at 29
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 226
  Illegal AgeI site found at 549
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 520
  Illegal SapI.rc site found at 667
  

Characterization

Verification of Part

The part was sequence verified in the pSB1C3 backbone before submission to the registry. Two reads, forward and reverse, were obtained using VF2 and VR (Figure 1).

Figure 1. This part was sequence verified. The silent mutations seen at the beginning of the coding region are from codon optimization that was not inputted into the reference sequence. There were three minor mutations that we do not expect to modify the function of the protein (hydrophobic to hydrophobic)

Results

This part was not investigated, but it could be verified using a basicity resistance assay.