Part:BBa_K2212003

GolS w/ pconII+RiboswitchF+DYKDDDDK+rrnBT1

This part contains coding sequence for enzyme inositol 3-alpha-galactosyltransferase (EC 2.4.1.123), 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 41
  Illegal AgeI site found at 367
  
• 1000
  COMPATIBLE WITH RFC[1000]

Gel Validation

The insertion is validated by enzyme digestion analysis with enzymes EcoRI and SacI, and CutSmart buffer.

The two clones highlighted have the expected ~1kbp and 2.4kbp bands

Figure 2: Gel image of the validation of GolS.

Sequencing

After gel validation, we sent one clone (clone #6, the first one from the right in the gel image above) for sequencing validation. The primer used were:
Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3'
Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'
The alignment results are shown below.

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