Team:OLS Canmore/Design

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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 the waste, incineration on site, the removal and transporting of waste as well as the formation of new low-quality 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 constructs. This was done by improving upon the Chicago and Sheffield iGEM teams’ previous projects, as they both previously worked with keratinases. The University of Chicago was unable to successfully express KerA in Bacillus due to issues that occurred during their transformation. The University of Sheffield iGem team had an unsuccessful expression of keratinase US due to cleaving issues that resulted from the differing signal peptides between gram-positive and gram-negative bacteria.

For our project, we decided to use E. coli JM109 and DH5alpha, which is gram-negative, as our bacterial chassis. The coding regions in the biobricks 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 the 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. We also decided to use an IPTG-inducible promoter (part BBa_J04500) for our expression cassette for this enzyme. The composite part was then synthesized into a pUC57 backbone, and using 3A assembly was placed into the standard biobrick pSB1C3 backbone.

Integration

While working on creating our construct, we began to create an implementation plan for our project within the poultry and wastewater industries. We furthered our research by coming in contact with various individuals in the poultry industry. Through our communication, we discovered that feather waste is dealt with by placing all of the waste in either a container to be dealt with. If it is not disposed of on site, it is transported in large quantities to be dealt with elsewhere. Because feathers are all handled with in a similar way, it was quite easy to foresee and plan the integration of the project in this industry. Since the feathers remain in the bioreactor to be degraded, we concluded that our keratinase expressing bacteria could be added to these feather holding containers. Not only would it be easy to pour in, but it would also have a lower environmental impact in comparison to the current incineration, burial, and mechanical degradation methods. In order to ensure the easy implementation of our project, our team came in contact with the Chicken Farmers of Canada, where they informed us about the lack of GMO regulations and that the implementation of our construct would not come in conflict with any bylaws.

After, we revisited the wastewater treatment facilities in order to learn about their processes. There, we noticed that both wastewater treatment facilities used bacteria somewhere within their processes. As a result, we believe that our project can be easily included in their system and strictly have positive effects. Due to conversations with our local wastewater treatment facility we were made aware that the particular part of the system that our project would be most effective in the early screening processes. This would be the best integration site for our project because it is where most of the keratin build up occurs. Whilst touring the various wastewater treatment plants within our community, we noticed that many of them utilized bioreactors or systems that worked very similarly.

As a result, we decided to design and construct our own prototype bioreactor as a way of demonstrating how our project can be implemented in both the poultry and wastewater treatment centres. Our prototype is a bubble column bioreactor that has been optimized to the specific conditions of E. coli, however we may need to consider a different chassis for implementation in the wastewater treatment industry. The bioreactor would be designed to contain our construct, as well as maintain the optimal conditions for effective growth and expression. This means that the prototype will allow our bacteria to survive at a temperature of 37 degrees Celsius and in media of a ph between 7 and 8. These conditions also align with the local wastewater treatment plants bioreactor, thus increasing the ease of application (see the page 'Demonstrate' for more information). For further development, we plan to create a sustainable business plan that can be used to distribute our construct to make it easier for companies to introduce our project within their facilities.

Benefits of Our Project

From all the research we have done, we believe that the use of a keratinase-expressing bacterial system will be a more effective way of dealing with keratin waste, as well as a more environmentally friendly option. One of the benefits for our construct opposed to other methods is how environmentally friendly it is. Methods like incineration and burial on site both contribute to to the growing greenhouse gas emissions problem. Not only does burial of waste aid in the release of greenhouse gases, it can also result in leachate. Unfortunately, these procedures only aim to remove keratin waste and don’t consider the detrimental impacts it has on the environment. Not only do we believe that our project will offer an environmentally conscious approach to degrading hair, but our team plans to take its use one step further through the creation of a useful protein product. Within the bioreactors, a sludge is formed as a byproduct of the degradation, and from our research throughout the year, we discovered that the sludge is formed by the same elements that make up fertilizers and animal feed. Once we completed our research on sludge, we finalized our integrative plan to collect the sludge inside a bioreactor and then ship it off to be created into useful protein products. After careful consideration, our team has come to the conclusion that the best way to collect the sludge would be through a skimming method. In contrast to the current processing, the implementation of our construct focuses on the recycling of material opposed to its removal. This aspect of our project is very important to our team because we wanted to not only develop a process that bypasses the harmful environmental effects, but also creates a nutrient-rich high-quality protein-product.

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