Team:OLS Canmore/Composite Part

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COMPOSITE PARTS

All the composite parts are composed of the same skeleton: A promoter, an R.B.S., a keratinase gene, and a double terminator. There is a composite part that uses the KerA gene, and one that uses the KerUS gene.

Part name: Keratinase A Expression Cassette (BBa_K1717000)
Creator: Our Lady of the Snows Catholic Academy 2016
Short description: Keratinase A (KerA) under control of a Lacl promoter

Long description: Keratinases are proteolytic enzymes that are capable of breaking the disulfide bonds in many different keratin substrates, such as hair and feathers. KerA is a basic part comprised of a synthesized KerA protein coding sequence, optimized for expression in E. coli and is intended for the breakdown of the beta-keratin, more commonly present in feathers.

KerA, when successfully expressed in cells, can be tested for enzymatic effectivity by looking for zones of clearing when plated on skim milk agar plates, as well observing the degradation of keratin substrates when grown with cells.

Design considerations: Keratinases are isolated from a number of bacteria and fungi species, most notably those in the Bacillus genera. The bacterial chassis we use is a strain of E. coli called K-12. Like all E. coli, this strain is gram-negative. Building off of the work of previous teams, such as Chicago, Sheffield, we deduced that the signal peptides that originated from gram-positive organisms could be causing the poor enzyme secretion in these teams projects. These teams either left these gram positive signals in their sequence, or removed it entirely. However, our team decided to replace the native signal peptide sequence and replaced it with one specifically optimized for secretion in gram-negative E. coli (pelB, with the sequence taken from part Part BBa_J32015). This part would hopefully secrete our enzyme into the periplasm, where it could escape into the media extracellularly once the promoter was induced. DNA sequences were compared using Clustal to ensure protein coding sequences were not altered, as well as to ensure that start and stop codons were correct. NEBCutter V2.0 was used to check for illegal cut sites. One EcoRI site and one PstI site were identified, and silent mutations were introduced in order to remove them.

Source: The sequence for the KerA coding region was taken from the Uniprot gene/protein sequence bank.


Part name: Keratinase US Expression Cassette (BBa_K1717173)
Creator: Our Lady of the Snows Catholic Academy 2016
Short description: Coding sequence for Keratinase US (KerUS)

Long description: Keratinases are proteolytic enzymes that are capable of breaking the disulfide bonds in many different keratin substrates, such as hair and feathers. Kerus is a basic part comprised of a synthesized Kerus protein coding sequence, optimized for expression in E. coli and is most active in breaking down alpha-keratin, more commonly found in hair.

Design considerations: Keratinases are isolated from a number of bacteria and fungi species, most notably those in the Bacillus genera. The bacterial chassis we use is a strain of E. coli called K-12. Like all E. coli, this strain is gram-negative. Building off of the work of previous teams, such as Chicago, Sheffield, we deduced that the signal peptides that originated from gram-positive organisms could be causing the poor enzyme secretion in these teams projects. These teams either left these gram-positive signals in their sequence, or removed it entirely. However, our team decided to replace the native signal peptide sequence and replaced it with one specifically optimized for secretion in gram-negative E. coli (pelB, with the sequence taken from part Part BBa_J32015). This part would hopefully secrete our enzyme into the periplasm, where it could escape into the media extracellularly once the promoter was induced. DNA sequences were compared using Clustal to ensure protein coding sequences were not altered, as well as to ensure that start and stop codons were correct. NEBCutter V2.0 was used to check for illegal cut sites. The gene contained no illegal cut sites.


Source: The sequence for the KERUS coding region was taken from the Uniprot gene/protein sequence bank.

Contact us at:
https://www.facebook.com/OLeSsence/
@igem_canmore
larvisais@redeemer.ab.ca