Part:BBa_K2739007
LacP-RBS-PhaP-HlyA-Terminator
This composite part is improved from the composite part of BBa_K390501, LacP-RBS-PhaP-HlyA-Terminator, which the Phasin part sequencing result revealed a stop codon. This improved composite part has the stop condon removed (See the design page for the sequence alignment).
Usage and Biology Phasins are low-molecular weight proteins,which possess an important role in PHB granule formation and can be found in microorganisms that naturally produce PHAs (Mezzina and Pettinari, 2016). Their rexpression can largely promote PHA production. This is a new part of the Phasin, which the previous available Phasin biobrick contain a stop codon. Due to its nature of binding to PHA granule surface, this protein could be engineered to fused with HlyA signal peptide, which would allow intracellular material (phasin and PHA)to be secreted out of the cell.
Result
The in-frame fusion transcript of LacP-RBS-PhaP-HlyA-Terminator (BBa_K390501), which generates an open reading frame in the mRNA and allow the translation of the fusion proteins, were mentioned on its iGEM registry that it might not be correct. Due to the importance of the Phasin-HlyA in the PHB secretion system, it was re-sequenced by Edinburgh Genomics.
The result revealed the correct sequence of the in-frame fusion transcript but suggested that there was a stop codon at the end of the Phasin sequence. A stop codon in the reading frame would prevent the expression of the fusion protein Phasin-HlyA and lead to the failure of the PHB secretion in the engineered E.coli. A PCR strategy was used to remove the stop codon, with the set of PCR primers designed by team member, Qihui Lian (Table 4). In addition, a HindIII restriction enzyme site was designed on the overhang of the forward primer to allow later confirmation of successful stop codon removal because this site is not present in the original LPH Biobrick.
Figure 1. Agarose gel electrophoresis of Lac promoter-Phasin-HlyA product. A. PCR product of Lac promoter-Phasin-HlyA with stop codon removal. B. Enzyme digestion of Lac promoter-Phasin-HlyA PCR product for stop codon removal (lane 1: 1 kb DNA ladder; 2: EcoR1 and HindIII double digestion) with the label of HlyA + pSB1C3 Backbone and Phasin.
The effect of stop codon removal on growth curve
In order to characterise the effect of LPH BioBrick with stop codon removal (LPH S-) to recombinant E.coli, the DNA plasmids of pSB1C3, LPH and LPH S- were transformed to E.coli BL21(DE3) strains and assessed their growth curve.
Figure 2. The growth study of recombinant E.coli BL21(DE3) strains in triplicate. Results are represented as the mean OD600nm ± S.E.M. The growth study of recombinant E.coli with pSB1C3, Lac promoter-Phasin-HlyA with and without stop codon.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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