Part:BBa_K390050

psbA2 promoter, 5'UTR, and RBS with GFP Protein generator shown to function in E. coli (Figure 1). Testing in Synechocystis sp. PCC 6803 pending.

Promoter is from psbA2 gene from Synechocystis sp. PCC 6803. The promoter region contains the promoter, 5'UTRand RBS of this operon. The promoter belongs to the Type I promoter family from Synechocystis sp. PCC 6803, and possesses a -30 box and a -10 box. The gene produces the reaction center protein of photosystem II (PSII). In PCC 6803, the promoter is recognized by sigma factors SigA and SigD, making it likely to be constituatively expressed through SigA, with modifications to transcription level influenced by light level through sSigD.

Protein generator was used as the standard for expression levels of USU iGEM 2010 team parts in E. coli. Expression results shown in Table 1 below. Expression in E. coli is due to the high homology with sigma70 promoter binding sequence and the E. coli consensus RBS sequence (Tables 2 and 3).

Figure 1. Cell plating of Synechocystis promoters driving GFP expression in E. coli.

Table 1.Promoter expression levels of Synechocystis sp. PCC 6803 promoters in E. coli as observed by USU 2010 iGEM team. All levels are shown relative to then psaAB promoter (BBa_K390049).

Table 2. Sigma factor binding site alignments. Underlined sequences are the binding region for sigma70 sigma factor from E. coli. Bases in red are exact matches to the consensus sequence.

Table 3. Ribosome binding site sequence alignments. The regions 25 bp upstream of the start codon for each gene were aligned to the E. coli consensus RBS sequence. Underlined sequences are the binding region for the 9 bp 3’ end of the 16S rRNA subunit in E. coli. Bases in red are exact matches to the consensus sequence.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 97
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 616
  Illegal XhoI site found at 517
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]