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<h4>As chloroform is extremely toxic and can have severe effects on the central nervous system. However, as chloroform is the only well established method for periplasmic protein extraction from our research ,we compromised its usage by increasing the stringency of our laboratory safety and minimised our usage. Minimal quantities of chloroform were used where possible and the entire USYD iGEM team were made aware of all the safety issues via safety forms, prior to handling the chemical. </h4> | <h4>As chloroform is extremely toxic and can have severe effects on the central nervous system. However, as chloroform is the only well established method for periplasmic protein extraction from our research ,we compromised its usage by increasing the stringency of our laboratory safety and minimised our usage. Minimal quantities of chloroform were used where possible and the entire USYD iGEM team were made aware of all the safety issues via safety forms, prior to handling the chemical. </h4> | ||
<h4><br>Due to the problems of chloroform for extraction of periplasmic insulin, our team focused on optimising Bacillus subtilis insulin production as it involved secretion into the media. Purification of insulin in Bacillus does not involve the need to lyse cell walls and only removal of media. This method would additionally be commercially ideal, particularly as our project design is to minimise the need for additional purification steps. The lack of need for chemicals like chloroform will have significant benefits, especially in large scale insulin production. </h4> | <h4><br>Due to the problems of chloroform for extraction of periplasmic insulin, our team focused on optimising Bacillus subtilis insulin production as it involved secretion into the media. Purification of insulin in Bacillus does not involve the need to lyse cell walls and only removal of media. This method would additionally be commercially ideal, particularly as our project design is to minimise the need for additional purification steps. The lack of need for chemicals like chloroform will have significant benefits, especially in large scale insulin production. </h4> | ||
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+ | <h6> RADIOACTIVE 14C IN HEPATOCYTE ASSAY </h6> | ||
+ | <h3>Issue: Use of 14C radioactive carbon to measure insulin bioactivity</h3> | ||
+ | <h2>Solution: Supervision and training</h2> | ||
+ | <h4> We tested the bioactivity of insulin by measuring the cell's uptake of radioactive glucose. Importantly, this is the 'gold standard' to test insulin in human and mouse cell lines so we decided to still use the assay instead of an alternative. We did however make sure we performed an ELISA first and only used successful results to ensure we minimised contact with radioactive material.<br><br> | ||
+ | Most importantly, we were supervised throughout the entire procedure by researchers who were experienced in these cell assays. Additionally, we used the smallest amount of radioactive glucose for our results to reduce any risks.</h4> | ||
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Revision as of 14:24, 1 November 2017
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Safety Design
Issue: Mouse Convulsion Testing
Solution: Assays using human tissue culture / ELISA assays
In testing whether our insulin produced was the correct structure and functioned in the correct way, we decided to avoid performing a mouse convulsion test which is the standard method used. In order to avoid the ethical issues surrounding induced murine fatality, we decided to test our insulin using an ELISA assay with an antibody specific to human insulin. This would determine whether our proinsulin had the correct three-dimensional structure.
As our single chain insulin will have a different structure, we do not expect it to interact with an insulin specific antibody. We will further test our insulin performing assays on human tissue culture – adipocyte tissue and/or pancreatic tissue. These assays were performed alongside insulin manufactured from different pharmaceutical companies to compare the response. These two assays will prevent the need to use live biological organisms. Importantly, using human cell lines will allow us to determine directly whether our single-chain insulin and proinsulin interacts with the correct insulin receptor in stimulating the correct response. An adipocyte assay will thus provide us with the same results as a mouse convulsion test without the same ethical issues.
Issue: Insulin can be fatal when injected
Solution: prevent handling of any needles and handling of small amounts
As insulin can actually cause fatality in humans, when administered to the body, we decided to avoid the potential of injecting insulin by avoiding needles completely in all of our assays. Our insulin was kept in culture for the majority of the time and when purified, was stored in sealed bottles All sharps were removed from the area in which we handled insulin.
As insulin is primarily lethal in high doses, we also minimised the handling of large amounts of insulin. However, as our project is focused on optimising the production of insulin, our initial assays before transferring to large scale fermentation involved small media cultures. This ensured that we were not handling dangerous levels of insulins at any point in time until our project design was validated.
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Safety Shipping
Are we going to have any issues with shipping?
As our biological parts that are constructed involves plasmids without any potential infectious particles eg. viral particles, we expect shipping to be very safe. Our constructs itself are already placed in the pSB1C3 vector that is standard in the iGEM registry, without a replicative origin. In order to prevent any additional issues, we also ensured all biological materials had double containment – containment in eppendorf tubes, additionally sealed using parafilm and/or clingwrap.
Specific to the experience of our laboratory, we found that in Australia our strict border control provided us with difficulties in obtaining our parts in the mail. With a bit of foresight and open communication, we could potentially mitigate this issue for the iGEM HQ.
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