Difference between revisions of "Team:OLS Canmore/Design"

DESIGN

This year, our team continued to build upon our previous year’s project in hopes of using synthetic biology to solve the keratin waste problem, in wastewater treatment facilities and the poultry industry. It was discovered that hair build up in the pipes of wastewater treatment facilities can lead to the damaging of equipment as well as cause processes to slow down.The current method of removing waste is done manually which is problematic as it is very expensive and time consuming.

Along with the issue of hair build up in wastewater treatment plants, we are also tackling the build up of feathers within the poultry industry. Because large quantities of feathers are produced, the current disposal methods are not very effective or environmentally conscious. Our team decided to fix these issues by effectively degrading the keratin within hair and feathers while simultaneously lowering the environmental impact. In order to counteract the keratin waste issue, our team has designed two keratinase expression cassettes, for expression in E. coli K12. The first keratinase expressed in E. coli we have synthesized is keratinase A (KerA), which is an enzyme optimized for the breakdown of hair. The second keratinase that was synthesized was keratinase US (KerUS), an enzyme optimized for feather degradation.

Research

In order for our project to have a useful application we need to ensure that its implementation is more effective than current methods of keratin waste degeneration. One of the companies that we contacted for assistance with our implementation strategy was Walker industries. Walker industries specializes in the creation of biosolids, meaning that they create high quality fertilizer from fecal matter and hair. Geoff Boyd, the general manager informed us that our construct would serve as a better use in the wastewater treatment plant over his industry. However, he mentioned that our project could help fix minor problems and he suggested to focus on implementing our project within wastewater treatment facilities and poultry industries. As a result, we continued to look at the current disposal processes for both feathers and hair. While researching, it was discovered that there are currently four ways of dealing with keratin waste. This consists of burial of waste, incineration on site, the removal and transporting of waste as well as the formation of new products. Once we were aware of the current methods of disposal, we began to research the benefits of keratinase more extensively.

Construct Creation

To begin the creation of our biobrick, our team had to start off by designing and optimizing our construct. This was done by improving upon the Chicago and Sheffield iGem team’s previous projects, as they both previously worked with keratinases. The University of Chicago was unable to successfully express KerUS in Bacillus due to issues that occurred during their transformation. The University of Sheffield iGem team had an unsuccessful expression of keratinase A due to cleaving issues that resulted from the differing signal peptides in gram positive and negative bacteria.

For our project, we decided to use a gram negative E.coli strain called DH5alpha as our bacterial chassis and our coding regions consisted of KerA and KerUS genes. These genes came from organisms in the Bacillus genera, which is gram positive. As we learnt from the University of Sheffield iGem team, this was quite problematic because the secretion of keratinase requires the “tagging” of a short amino acid signal. This signal is required to give it folding instructions to prevent the misfolding of the enzyme within the periplasm. The signal will also allow the secretion tag to be removed, thus resulting in the expression of our proteolytic enzymes. Because of the signal sequence differentiation between gram positive and gram negative bacteria, we had to optimize the gene sequences by changing the export tag. With the help of our mentors, we decided to completely remove the gram positive signal and replace it with a signal peptide called pelB. The composite part was then synthesized into the standard biobrick format on a psb1C3 backbone. We also decided to use an IPTG-inducible promoter (part BBa_J04500) for our expression cassette for this protein.