Difference between revisions of "Team:ColumbiaNYC/Applied Design"

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     <h2>Gentamicin Assay</h2>
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     <h2>
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      The Advantages to a Synthetic Biology Approach
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      The next step to proving the therapeutic efficacy of the presented mechanism is to ensure that the bacteria are actually
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        A cornerstone in synthetic biology is the connection of distinct biological functions to create useful system
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      entering their target cells and delivering the shRNA payload to them. The way in which to do this is a gentamicin assay.
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        level behavior. We can take advantage of how bacteria naturally localize in tumors and mass produce products, invade cells,  
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      A gentamicin assay allows us to see whether the bacteria have entered mammalian cells. Essentially how this is done
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        and release the products directly into the cells. This allows easily-degraded
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      is that we leave the cells exposed to the bacteria for a given amount of time, then apply gentamicin to the cells,
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        compounds to be effectively delivered and prevents these compounds from affecting healthy
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      killing off any external bacteria. Then we allow for incubation and infection and observe the progression of the invasion
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        cells. The combination of these mechanisms creates a powerful, cancer-specific circuit for gene therapy.
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      using microscopy over the course of 24 hours. This will indicate whether the bacteria have truly invaded the cells.
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     <h2>Mouse Models</h2>
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      Expanding the Possibilities of Gene Therapy
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       After the gentamicin assay, another important step in this project will be mouse models. We need mouse models to test the
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       Anticipating the applications of synthetic biology to healthcare, we devised a therapeutic approach to modulate mammalian
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       efficacy of this system in vivo, assess toxicity adequately, and ensure that the bacteria can reach their target cancer
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      gene expression at the post-transcriptional level. Recombinant E. coli with the capacity to invade mammalian cells
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       sites specifically. A study of the delivery of our shRNA containing bacteria to tumors in live mice would be the final
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       will deliver an shRNA payload against an aberrantly expressed gene, for example an oncogene in cancer or proinflammatory
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       step in ensuring the therapetic relevance of this mechanism.
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       cytokine in inflammation, to the host cytoplasm. This shRNA payload will then inhibit protein function, which can combat
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       mutations that confer resistance to traditional therapies, as with the tyrosine kinase inhibitors gefitinib and imatinib.
 
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     <h2>Lipofectamine</h2>
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      The Future Outlook
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       eGFP HeLa cells were grown in Dulbecco’s Modified Eagle Media until they had reached 80% confluence. They were then split,
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       Our mechanism to modulate mammalian gene expression can have a variety of applications, extending throughout as well beyond
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       and aliquotted into 12 individual wells. These cells were then incubated overnight. In the mean time, bacteria containing
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       healthcare. The engineered bacteria would have a significant application to human health particularly in conditions
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       the eGFP shRNA were induced to produce this shRNA, which was then isolated with an miRNA mini prep kit. After this,
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       characterized by aberrant gene expression, such as with oncogenes in cancer, cytokines in inflammation, and many others.
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      concentrations of shRNA were determined via nanodrop, and a gel was run to ensure that our desired shRNA were present.
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       For cancer applications, the engineered bacteria can be taken as an oral probiotic, which will then selectively localize
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       The shRNAs were then allowed to complex with lipofectamine 2000, producing complexes containing the desired shRNA.
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       in tumor cells to prove gene therapy.
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      The HeLa cells were then exposed to these complexes. Three wells were not exposed to anything, another three were exposed
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      to just liposomes, the next three were exposed to liposomes with control eGFP siRNA, and the final three wells were
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      exposed to liposomes containing eGFP shRNA. The cells were allowed to incubate for 24 hours, after which they were
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       viewed under a microscope and subjected to flow-cytometry.
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    <p>
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      Lipofectamine results: The results showed that eGFP siRNA knockdown of eGFP expression in HeLa cells was less than that of
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      shRNA knockdown of eGFP but greater than the control indicating that the method of gene knockdown is in-fact a viable
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      method.
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Revision as of 22:42, 1 November 2017

Applied Design

Our design applied to a real world problem

The Advantages to a Synthetic Biology Approach

A cornerstone in synthetic biology is the connection of distinct biological functions to create useful system level behavior. We can take advantage of how bacteria naturally localize in tumors and mass produce products, invade cells, and release the products directly into the cells. This allows easily-degraded compounds to be effectively delivered and prevents these compounds from affecting healthy cells. The combination of these mechanisms creates a powerful, cancer-specific circuit for gene therapy.

Expanding the Possibilities of Gene Therapy

Anticipating the applications of synthetic biology to healthcare, we devised a therapeutic approach to modulate mammalian gene expression at the post-transcriptional level. Recombinant E. coli with the capacity to invade mammalian cells will deliver an shRNA payload against an aberrantly expressed gene, for example an oncogene in cancer or proinflammatory cytokine in inflammation, to the host cytoplasm. This shRNA payload will then inhibit protein function, which can combat mutations that confer resistance to traditional therapies, as with the tyrosine kinase inhibitors gefitinib and imatinib.

The Future Outlook

Our mechanism to modulate mammalian gene expression can have a variety of applications, extending throughout as well beyond healthcare. The engineered bacteria would have a significant application to human health particularly in conditions characterized by aberrant gene expression, such as with oncogenes in cancer, cytokines in inflammation, and many others. For cancer applications, the engineered bacteria can be taken as an oral probiotic, which will then selectively localize in tumor cells to prove gene therapy.