Team:OLS Canmore/Basic Part
BASIC PARTS
Keratinases are a type of proteolytic enzymes, meaning they are capable of breaking down proteins into smaller units (1). While keratinases have been isolated within several different fungal and bacterial species, they have not been commonly expressed in E. coli K12 which is a gram-negative bacteria.
For the project we are using a strain of E. coli called JM109 as our bacterial chassis which allows us to express protein at a relatively high rate and to work more easily in the lab.
Keratinases were previously used by the Chicago and Sheffield iGEM teams. Chicago attempted to express keratinase in Bacillus subtilis but was unsuccessful in transforming the genes. The University of Sheffield experienced a cleaving issue as they did not remove the signal peptide from the Bacillus (gram-positive) gene sequence. This causes an issue because of the differing amount of membranes between gram-positive and gram-negative bacteria, and the differences in how the bacteria must secrete the enzymes. Therefore, in optimizing the keratinase genes for expression in E.coli, we removed the gram-positive signal sequence entirely and replaced it with pelB (a signal peptide, and biobrick part, optimized for secretion of protein in E.coli).
Figure 1: Shows a generalized depiction of the membrane differences between Bacillus and E.coli. The light blue tag at the end of the protein represents the native signal peptide, while the orange tag is representative of the optimization. During the optimization, the original signal peptide was completely removed and replaced with pelB.
Once these basic part are paired with a promoter, keratinase should be expressed and secreted into the periplasm. Because of the toxicity of accumulating high amounts of keratinase, the bacteria would the release keratinase from the periplasm into the cell-growth media.
These parts were synthesized and placed in the pSB1C3 backbone using the standard biobrick assembly. Before the synthesis, the correct start and stop codons were added, and we checked to ensure that the protein coding sequences were not altered during the other optimization steps by comparing the DNA sequences using Clustal. NEBCutter V2.0 was also used to check for any illegal restriction sites. Keratinase A (KerA) contained two illegal cut sites, but a silent mutation was introduced to solve the issue. Keratinase US (KerUS) did not contain any.
The genes optimized for expression in E.coli and in standard biobrick format are the sequences listed in the parts pages on the registry.
Keratinase A (BBa_K1717171)
Keratinases are proteolytic enzymes which mainly break the disulphide bonds in various keratin substrates. KerA is a basic part comprised from a synthesized KerA protein coding sequence, optimized for expression in E. coli and most active in degrading keratin in feathers. This gene, when expressed in cells, can be tested for activity through looking for zones of clearing on skim milk agar plates where cells are growing, as well as by looking at degradation of keratin-containing substrates (feathers, hair, chemically synthesized keratin, etc.) when grown with cells.
Keratinase US (BBa_K1717172)
Keratinases are proteolytic enzymes which mainly break the disulphide bonds in various keratin substrates. KerUS is a basic part comprised of a synthesized KerUS protein coding region, optimized for expression in E. coli and most active in degrading keratin in hair. This gene, when expressed in cells, can be tested for activity through looking for zones of clearing on skim milk agar plates where cells are growing, as well as by looking at degradation of keratin-containing substrates (hair, feathers, chemically synthesized keratin, etc.) when grown with cells.
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@igem_canmore
larvisais@redeemer.ab.ca
