Part:BBa_K2212004

GLA w/ pconII+RiboswitchF+DYKDDDDK+rrnBT1

This part contains coding sequence for enzyme alpha-galactosidase (EC 3.2.1.22), 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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 526
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 415
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

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

  Illegal BsaI.rc site found at 380
  

Gel Validation

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

The two highlighted clones have the expected ~1.3kbp band

Figure 2: Gel image of the validation of GLA.

Sequencing

After gel validation, we sent one clone (clone #9, the highlighted one on 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 4: Sequencing validation for GLA insertion. 3' end.