Team:SUIS Shanghai/Parts

Parts

Composite Part

Our main construct, the composite part BBaK269000 contains five Open Reading Frames (ORFs), from pvsA to pvsE that when together, form a gene cluster which synthesizes and secrete the siderophore vibroferrin. This part is composed of 5 ORFs Among the cluster, pvsB and pvsD contribute for bond formation between 2-ketoglutarate and l-alanine, or L-alanine and ethanolamine. PvsA is thought to form ester bond between citrate and ethanlamine. PvsE, catalyzes decarboxylation from an amino acid to ethanolamine or its precursor. In addition to the four coding sequences for the synthesis of vibrioferrin, our composite part also contains the coding sequence pvsC, which most likely a gene which codes for a transporter that secretes the final product out of the cell. (Tanabe et al., (2003); and Fujita et al., (2011))

Our ORFs are controlled by the string T7 Promoter part BBa_I712074 so that we can induce the coding sequences and over-express the gene cluster under optimal conditions. With induction with IPTG, lac repressor would fall of Lac operator on BL21 cells chromosomal genome, the T7 polymerase would be transcribed and translated that could be bind to T7 promoter on our pET plasmid. Then our gene of interest which is from pvsA to pvs E would be transcribed and translated. Vibroferrin would be produced, and secreted outside e.coli through pvsC. The double terminator sequence composed of the parts B0010 and B0012 end transcription. Ribosome binding site part J61101 was used in between each ORF in order to ensure translation of each coding sequence and thus, our system will produce the enzymes required for biosynthesis and secretion of vibrioferrin.


Parts Table


Part Name Part Description
BBa_K2690000 T7 Promoter + Vibrioferrin Biosynthesis Gene Cluster + Double Terminator
BBa_K2690001 pvsA - Vibrio parahaemolyticus vibrioferrin synthesis gene A
BBa_K2690002 pvsB - Vibrio parahaemolyticus vibrioferrin synthesis gene B
BBa_K2690003 pvsC - Vibrio parahaemolyticus vibrioferrin synthesis gene C
BBa_K2690004 pvsD - Vibrio parahaemolyticus vibrioferrin synthesis gene D
BBa_K2690005 pvsE - Vibrio parahaemolyticus vibrioferrin synthesis gene E

Basic Parts & Primers


In addition to our composite part, which is essential for the functioning of our whole device, we documented each ORF corresponding to each gene sequence with the cluster. Descriptions of the functions of each gene can be found below as well as primers we designed for the amplification of each sequence and Biobrick construction (overhangs for prefix and suffix addition).


PvsA

  • Forward Primer
  • 5’ – GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAATCTACGATGATTATCG – 3’
  • G-C content = 44.0%; Tm = 68.5 C
  • Reverse Primer
  • 5’ – GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTATGCTGCCACCTCCTCGCGAC– 3’
  • G-C content = 54.4%. Tm = 74.3 C


PvsB

  • Forward Primer
  • 5’ –GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAATCACAACGCTTTATACCTAACC– 3’
  • G-C content = 45.5%; Tm = 70.4 C
  • Reverse Primer:
  • 5’ – GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAACGTACGCTTTTTTTGATGGC – 3’
  • G-C content = 42.6%; Tm = 69.1 C


PvsC

  • Forward Primer
  • 5’ – GTTTCTTCGAATTCGCGGCCGCTTCTAGATGACCAAACTCATCTACAGTGTG – 3’
  • G-C content = 48.1%; Tm = 70.8 C
  • Reverse Primer:
  • 5’ –GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGTCTGAGAGGATTGAGTTTGTG– 3’
  • G-C content = 45.6%; Tm = 70.0 C


PvsD

  • Forward Primer
  • 5’ – GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTAACAAACTCAATCCCAGA – 3’
  • G-C content = 47.1%; Tm = 70.3 C
  • Reverse Primer:
  • 5’ – GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGACATGTTGCGCTCCTTTTTCG– 3’
  • G-C content = 47.4%; Tm = 71.7 C


PvsE

  • Forward Primer
  • 5’ – GTTTCTTCGAATTCGCGGCCGCTTCTAGATGTCTAATTCACTCTCCCTAC– 3’
  • G-C content = 48.0%; Tm = 69.8 C
  • Reverse Primer:
  • 5’ – GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGAAATAGTGGAAGATCGCCGGTTC– 3’
  • G-C content = 47.5%; Tm = 71.4 C



References:

Tanabe, Tomotaka, Funahashi, Tatsuya, Nakao, Hiroshi, Miyoshi, Shin-Ichi, Shinoda, Sumio, & Yamamoto, Shigeo. (2003). Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus. The Journal of Bacteriology, 185(23), 6938-6949.
Fujita, M., Kimura, N., Sakai, A., Ichikawa, Y., Hanyu, T., & Otsuka, M. (2011). Cloning and heterologous expression of the vibrioferrin biosynthetic gene cluster from a marine metagenomic library. Bioscience, Biotechnology, and Biochemistry, 75(12), 2283-7.

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