Part:BBa_K2212002

RafS w/ pconII+RiboswitchF+DYKDDDDK+rrnBT1

This part contains coding sequence for enzyme Raffinose synthase (EC 2.4.1.82), pconII promoter, riboswitch F, DYKDDDDK-tag, rrnB T1 terminator. The unlabeled sequences are random filler sequences. The protein sequence is originated from Arabidopsis thaliana. The protein coding sequence was codon optimized for Synechocystis sp. PCC 6803.

This part is intended to be used in Synechococcus elongatus PCC 7942 in the pathway for the production of tagatose from sucrose. Originally, this protein is an important enzyme in plant galactose metabolism pathway.

Figure 1: Proposed tagatose synthesis pathway.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 410
  Illegal BamHI site found at 1471
  Illegal XhoI site found at 527
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 41
  Illegal AgeI site found at 2020
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 564
  Illegal BsaI.rc site found at 429
  

Gel Validation

Primers:
Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3'
Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'

All three clones have the expected ~2.2kbp band

Figure 2: Gel image of the validation of RafS.

Sequencing

After gel validation, we sent one clone (clone #13, the first one from the left in the gel image above) for sequencing validation. The primers used were the same ones as used for PCR (Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3' Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'). The alignment results are shown below.

Figure 3: Sequencing validation for RafS insertion. 5' end.

Figure 4: Sequencing validation for RafS insertion. 3' end.