Part:BBa_K2212005

LAI w/ pconII+RiboswitchF+DYKDDDDK+rrnBT1

This part contains coding sequence for enzyme L-arabinose isomerase (EC 5.3.1.4), 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 that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose.

Figure 1: Proposed tagatose synthesis pathway.

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 1267
  Illegal NgoMIV site found at 1433
  Illegal AgeI site found at 41
  Illegal AgeI site found at 1366
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 374
  

Gel Validation

We used PCR to validate the insertion of the part.
Primers:
Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3'
Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'

All three clones have the expected ~1.5kbp band

Figure 2: Gel image of the validation of L-AI.

Sequencing

After gel validation, we sent one clone (clone #15, the highlighted one in the middle 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 L-AI insertion.