Team:Lethbridge Canada/The Project

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Mlc is a repressive protein that binds to a specific DNA sequence and prevents it from being transcribed. This regulates the transcription of specific genes so certain proteins are only made when they are needed. This is the mechanism we are going to use to regulate the transcription of the gene for insulin. When glucose enters the cells via enzyme II, it picks up a phosphoryl group that was originally bound to EIICBGlc. The dephosphorylated form of EIICBGlc has a high affinity with Mlc and recruits MCL from its binding site on the DNA sequence. The gene is now free to be transcribed. Due to the fact that this sequence is originally stimulated by an increase in glucose concentration as it enters the cell, it is the perfect circuit for us to use to regulate the production of inulin.
Mlc is a repressive protein that binds to a specific DNA sequence and prevents it from being transcribed. This regulates the transcription of specific genes so certain proteins are only made when they are needed. This is the mechanism we are going to use to regulate the transcription of the gene for insulin. When glucose enters the cells via enzyme II, it picks up a phosphoryl group that was originally bound to EIICBGlc. The dephosphorylated form of EIICBGlc has a high affinity with Mlc and recruits MCL from its binding site on the DNA sequence. The gene is now free to be transcribed. Due to the fact that this sequence is originally stimulated by an increase in glucose concentration as it enters the cell, it is the perfect circuit for us to use to regulate the production of inulin.
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Nam, T., Cho, S., Shin, D., Kim, J., Jeong, J., Lee, J., Roe, J., & Kang, S. (2001). The escherichia coli glucose transport enzyme iicb(glc) recruits the global repressor mlc. Retrieved from http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=historysearch&querykey=1
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Nam, T., Cho, S., Shin, D., Kim, J., Jeong, J., Lee, J., Roe, J., & Kang, S. (2001). The Escherichia coli glucose transport enzyme iicb(glc) recruits the global repressor mlc. Retrieved from http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=historysearch&querykey=1

Revision as of 23:20, 13 June 2012

Construct.JPG
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Project

Diabetes
The purpose of our project is to create bacteria that will detect blood sugar levels and respond accordingly by producing insulin. Type one diabetes is caused by the degeneration of islet cells in the pancreas. Conventional methods of treatment for type one diabetes include direct injection of insulin intravenously, the transplantation of islet cells or even the introduction of an entirely new pancreas. Our engineered bacteria would provide a long-term solution compared to the standard injections, which need to be administered at least twice per day. In essence, our project has the potential to change the way that diabetes is treated.


Glucose Detection
It is essential that our project must include a way for Escherichia coli to detect changing glucose concentrations in its environment. If the transcription of the insulin gene is not regulated in some way then the protein will be constantly produced which will lead to an extremely high concentration of insulin in the system, which can be just as dangerous as high blood sugar levels.

Sensory transduction is a mechanism used by many organisms to monitor their external environments. E. coli detects changes in glucose levels in its environment by means of phosphotransferase system (PTS). This system uses E. coli’s enzyme II, which consists of the membrane-bound protein EIICBGlc, as well as enzyme I (EI), a histidine phosphocarrier protein HPr, and another protein called EIIAGlc. Enzyme II is responsible for the phosphorylation and transportation of glucose into the cell.

Mlc is a repressive protein that binds to a specific DNA sequence and prevents it from being transcribed. This regulates the transcription of specific genes so certain proteins are only made when they are needed. This is the mechanism we are going to use to regulate the transcription of the gene for insulin. When glucose enters the cells via enzyme II, it picks up a phosphoryl group that was originally bound to EIICBGlc. The dephosphorylated form of EIICBGlc has a high affinity with Mlc and recruits MCL from its binding site on the DNA sequence. The gene is now free to be transcribed. Due to the fact that this sequence is originally stimulated by an increase in glucose concentration as it enters the cell, it is the perfect circuit for us to use to regulate the production of inulin.

Nam, T., Cho, S., Shin, D., Kim, J., Jeong, J., Lee, J., Roe, J., & Kang, S. (2001). The Escherichia coli glucose transport enzyme iicb(glc) recruits the global repressor mlc. Retrieved from http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=historysearch&querykey=1



Insulin Production and Secretion
This glucose sensitive promoter will be coupled to the DNA for the production of the 51 amino acid polypeptide insulin (human insulin). Insulin needs to be produced and then exported out of the cell in order for the insulin to decrease the blood glucose levels. For the export of insulin, we plan to use a signal sequence that directs the cell to transport the insulin protein outside of the cell. This can be fused to the DNA that is in charge of the insulin production.


Insulin construct 044.jpg
Insulin Construct


Delivery System
The method of delivery of our organism can be through direct delivery in the bloodstream or through the NASA Bio capsule. It is feasible to use a system that enters the blood stream because there are already millions of E. coli cells in our body, and we can use a strain that does not cause an immune response. Alternatively, the NASA Bio capsule, which is a tangle of carbon nanotubes that will be used to contain particular cells and eventually medicinal substances inside it, could also be used. The capsule could contain our cells, and, should the body need insulin, automatically start secretion. The Bio capsule is tiny, inserted into the skin, non-reactive and fast acting.