BerkiGEM2007Present5

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'''This is a rough ROUGH draft. Excuse my spelling and grammar errors for now please!'''
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===Growth Control===
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  <p><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
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  <p><a href="https://2007.igem.org/BerkiGEM2007Present3">&lt;&lt;Previous Section: Controller</a> | <a href="https://2007.igem.org/BerkiGEM2007Present2">Next Section: Freeze Drying&gt;&gt;</a></p>
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----
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<h1 align="center">Growth Control</h1>
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Growth control in our system is established by the incorporation of a plasmid that can be triggered to translate a toxin. The toxins are endonucleases or rnases that destroy the genetic material within a bacteria and thus prevent the bacteria from replicating. Throughout the summer, we worked on several systems that would accomplish the goal of growth regulation: <br>
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<p align="justify"> To prevent chance of infection or unwanted proliferation after  hemoglobin production, we have engineered a genetic self-destruct  mechanism whereby when induced, the bacterial cell will express a  genetic material-degrading toxin which kills the cell, but leaves it  physically intact. Growth control in our system is established by the incorporation of a plasmid that can be triggered to translate a toxin. The toxins are endonucleases or RNAses that destroy the genetic material within a bacteria and thus prevent the bacteria from replicating. Throughout the summer, we worked on several different constructs of an inducible toxin  and screened for the ones with the best phenotype. </p>
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<p align="center">&nbsp;</p>
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******'''An Inducible Toxin''' <br>
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<h2 align="center"><strong>An Inducible Toxin</strong> </h2>
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******'''A Disruptable Equilibrium Between an Endonuclease and a Methylase''' <br>
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<p align="justify"> Using a PBAD promoter, we constructed several variations of inducible toxins, including the colicin DNAse CeaB, endonucleases BamHI and BglII, and RNAse barnase. Additionally, ribosome binding site libraries were used in order to increase the likelihood of finding a construct that would exhibit no growth after being induced with arabinose, but normal growth when uninduced. </p>
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******'''A Cre Recombinational Growth Switch'''''' <br>
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<p align="justify">We screened libraries of potential hits with a Tecan growth assay, and  in the end, the constructs that showed the desirable phenotype are the constructs shown below (I716408C and I716462). When uninduced, the cells show growth comparable to regular DH10B cells, but when induced, the growth plateaus as the cells lose their ability to replicate due to RNA and DNA destruction.</p>
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<p align="center"><strong>I716408C:</strong><img src="https://static.igem.org/mediawiki/2007/3/3b/Berk-Figure-Barnase.png" alt="" name="" width="298" height="156" align="middle"></p>
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<p align="center"><strong>I716462:</strong><img src="https://static.igem.org/mediawiki/2007/3/38/Berk-Figure-BamHI.png" alt="" name="" width="299" height="125" align="middle"></p>
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<p align="justify">&nbsp;</p>
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<p align="justify">&nbsp;</p>
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<h2 align="center">Characterization of Growth Ability</h2>
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'''Introduction''' <br>
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<p align="justify">Additional characterization of the systems include a titer experiment,  where the cells were grown up in a cultures both with and without  arabinose. The cultures were then diluted and plated such that the number of colonies could be counted. It was shown that the cultures  induced with arabinose had significantly fewer colonies than those not  induced with arabinose, after taking into account the various  dilutions.</p>
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Safety blah blah. In essence, we want our cells to be a sac filled with hemoglobin.
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<p align="center"><img src="https://static.igem.org/mediawiki/2007/9/9c/Berk-KillSwitchTiterdata.jpg" alt="" name="" width="827" height="639" align="middle"></p>
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<p align="justify">&nbsp;</p>
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<h2 align="center">Phenotype of dead cells</h2>
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'''An Inducible Toxin''' <br>
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<p align="justify">Importantly, although the cells triggered with arabinose have lost  their ability to reproduce, they do not lyse, because if the cells did  not remain intact, then they could not serve the purpose of  transporting hemoglobin. Equally significant is the fact that the the  proteins within the cell are not degraded and still maintain their  function.</p>
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Using a pBad promoter, we constructed several variations of inducible toxins, including the dnase ceaB from the coilicin activity protein, endonucleases BamHI and BglII, and rnase barnase. Additionally, ribosome binding site libraries were used in order to increase the likelihood of finding a construct that would exhibit no growth after being induced with arabinose, but normal growth when uninduced. <br>
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<p align="center"><img name="" src="https://static.igem.org/mediawiki/2007/b/b2/Berk-deadcellphenotype.jpg" width="482" height="186" alt=""></p>
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In the end, the construct that showed the desirable phenotype is the construct shown in the picture below. When uninduced, the cells showed growth comparable to regular DH10B cells, but when induced, the growth plateaus as the cells lose their ability to replicate due to RNA and DNA destruction.  
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<p align="justify">This phenotype was confirmed by transforming an RFP expression plasmid  into the cell and then taking microscopy pictures of the cells grown up  in cultures with and without arabinose. With microscopy, it is clear  that the cells are still capable of expressing the RFP protein even  when their growth has been arrested.</p>
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[[Image:pbadbarnasepic.jpg]]<br>
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<p align="center"><img name="" src="https://static.igem.org/mediawiki/2007/4/4a/Berk-killswitchmicroscopy1.jpg" width="626" height="487" alt=""></p>
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*Note to self - adjust the graph such that it shows the control of just regular DH10B
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<p align="center">&nbsp;</p>
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*Also include microscopy images
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<p align="center"><img name="" src="https://static.igem.org/mediawiki/2007/9/90/Berk-killswitchmicroscopy2.jpg" width="626" height="476" alt=""></p>
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<p align="center"><a href="https://2007.igem.org/Berkeley_UC">&lt;&lt;&lt; Return to UC Berkeley iGEM 2007 </a></p>
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<p align="center"><a href="https://2007.igem.org/BerkiGEM2007Present3">&lt;&lt;Previous Section: Controller</a> | <a href="https://2007.igem.org/BerkiGEM2007Present2">Next Section: Freeze Drying&gt;&gt;</a></p>
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'''Cre Recombination Model''' <br>
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<p align="justify">&nbsp;</p>
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*Still working on this!
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*Knocking it into the genome
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*I'm going to work my butt off until this works
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Latest revision as of 02:28, 27 October 2007

Untitled Document

<<< Return to UC Berkeley iGEM 2007

<<Previous Section: Controller | Next Section: Freeze Drying>>

Growth Control

To prevent chance of infection or unwanted proliferation after hemoglobin production, we have engineered a genetic self-destruct mechanism whereby when induced, the bacterial cell will express a genetic material-degrading toxin which kills the cell, but leaves it physically intact. Growth control in our system is established by the incorporation of a plasmid that can be triggered to translate a toxin. The toxins are endonucleases or RNAses that destroy the genetic material within a bacteria and thus prevent the bacteria from replicating. Throughout the summer, we worked on several different constructs of an inducible toxin and screened for the ones with the best phenotype.

 

An Inducible Toxin

Using a PBAD promoter, we constructed several variations of inducible toxins, including the colicin DNAse CeaB, endonucleases BamHI and BglII, and RNAse barnase. Additionally, ribosome binding site libraries were used in order to increase the likelihood of finding a construct that would exhibit no growth after being induced with arabinose, but normal growth when uninduced.

We screened libraries of potential hits with a Tecan growth assay, and in the end, the constructs that showed the desirable phenotype are the constructs shown below (I716408C and I716462). When uninduced, the cells show growth comparable to regular DH10B cells, but when induced, the growth plateaus as the cells lose their ability to replicate due to RNA and DNA destruction.

I716408C:

I716462:

 

 

Characterization of Growth Ability

Additional characterization of the systems include a titer experiment, where the cells were grown up in a cultures both with and without arabinose. The cultures were then diluted and plated such that the number of colonies could be counted. It was shown that the cultures induced with arabinose had significantly fewer colonies than those not induced with arabinose, after taking into account the various dilutions.

 

Phenotype of dead cells

Importantly, although the cells triggered with arabinose have lost their ability to reproduce, they do not lyse, because if the cells did not remain intact, then they could not serve the purpose of transporting hemoglobin. Equally significant is the fact that the the proteins within the cell are not degraded and still maintain their function.

This phenotype was confirmed by transforming an RFP expression plasmid into the cell and then taking microscopy pictures of the cells grown up in cultures with and without arabinose. With microscopy, it is clear that the cells are still capable of expressing the RFP protein even when their growth has been arrested.

 

<<< Return to UC Berkeley iGEM 2007

<<Previous Section: Controller | Next Section: Freeze Drying>>

 

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