Team:Calgary/Project/Future Directions

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<p><b>Reporter</b>: We would like to further characterize our reporter system, especially with regards to the optimal buffer solution that a sample should be placed in. Afterwards, we could test a variety of substrates with <i>lacZ</i> as well as other genes to determine what the optimal reporting molecule is. Also, seeing if we can develop a smaller and cheaper version of the setup is an option we would like to explore.</p>
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<p><b>Reporter</b>: Our next step is to start creating a standard curve in a calcium chloride buffer solution, supplemented with magnesium ions for beta-galactosidase. After this, we will create another curve using cells induced to produce beta-galactosidase as an <i>in vivo</i> assay in our new buffer system. After this, we will move on to testing these curves in tailings pond samples.</p>
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<br><p><b>qRT-PCR</b>: On the RT-PCR side of the promoter search there are a few more points to test.  Although we have identified a potential candidate gene, up-regulated in the presence of NA's, the next step is to test if the response observed can be duplicated with a general stress, such as hydrogen peroxide. Another thing to test is the specificity of the observed response, to see if structurally variant naphthenic acids can elicit the same response.</p><br>
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<br><p><b>qRT-PCR</b>: We are in the process of biobricking two promoter elements in addition to the submitted part and performing more electrochemical assays to test the reporter system in the presence of naphthenic acids. Future characterization experiments would explore enrichment of opposite strand transcripts of the fad-2 operon. Lastly, there were other candidate genes to investigate by bioinformatics screen that we were unsuccessful in developing PCR primers for. We are interested in sequencing the environmental isolate strains LD1 and LD2 to refine our screen and perhaps be able to design better primers for this line of investigation.</p><br>
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<p><b>"Sensory Element Fishing"</b>: For this side of the project we need to optimize the interaction between the streptavidin beads and our biotinylated compound. To do this we are going to have to find a way to efficiently separate our compound from the solution that it is currently in without damaging the biotinylated compound. After this is done we will be able to perform the IPs that we have envisaged.</p><br>
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<p><b>"Sensory Element Fishing"</b>: With our preliminary pull-down result, the next step is to repeat these several times in order to confirm any interactors that we are seeing.  We also wish to biotinylate more naphthenic acid-like compounds in order to create a biotinylated library of naphthenic acid molecules to further our search for sensory elements. We also want to expand our "fishing" model to search for other types of sensory elements (i.e. ChIP-SEQ).</p><br>
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<p><b>Chassis</b>: In terms of tools, further chaacterization of the HspA70/RbcS2 promoter and algal luciferase is needed. The addiiton of a few more selectable markers suitable for use in algae. Ultimately, we also want to finish the construction and screening of chloroplast and nuclear promoter libararies in <i>Dunaliella tertiolecta</i>. </p><br>
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<p><b>Chassis</b>: In terms of tools, further characterization of the <i>HspA70/RbcS2</i> promoter and algal luciferase is needed. The addition of a few more selectable markers suitable for use in algae would be useful. Ultimately, we also want to finish the construction and screening of chloroplast and nuclear promoter libararies in <i>Dunaliella tertiolecta</i>.  Now that our conjugation system is up and working, we also want to start attempting to conjugate our putative system (BBa_K640006) from <i>E. coli</i> and into <i>Pseudomonas</i>. </p><br>
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<p><b>Prototype</b>: The next step would be to miniaturize our circuit and create a printed circuit board. Printed circuit boards have the advantage of integrated wires, meaning the wires are small copper lines on the board instead of physical wires sticking up from the board.  We will also improve the software interface that controls our potentiostat.  <br>
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<p><b>Prototype</b>: During the development of our electrochemical reporter system we spent a lot of time thinking about what hardware could be used and found out that a potentiostat is the best option. A potentiostat is the electronic hardware needed to control the three electrode system used to measure the data in our experiments. It keeps the working electrode at a constant potential with respect to the reference electrode by adjusting and measuring the current between the working electrode and counter electrode. We obtained our data using a professional lab grade potentiostat, but we also spent some time looking into how they are designed and managed to built our own prototype. While it is built on a breadboard and is bigger than the potentiostat we used for the measurements, it is a valid proof of concept that potentiostats can be built using electrical circuit components no more complicated than an operational amplifier. <br>
 
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The ultimate goal would be to construct a fully functional prototype which could send the collected data directly to a computer. However, building a prototype was a lower priority compared to conducting the actual experiments and we weren’t able to finish it over the summer.
 
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Latest revision as of 03:54, 29 October 2011


Future Directions

Reporter: Our next step is to start creating a standard curve in a calcium chloride buffer solution, supplemented with magnesium ions for beta-galactosidase. After this, we will create another curve using cells induced to produce beta-galactosidase as an in vivo assay in our new buffer system. After this, we will move on to testing these curves in tailings pond samples.


qRT-PCR: We are in the process of biobricking two promoter elements in addition to the submitted part and performing more electrochemical assays to test the reporter system in the presence of naphthenic acids. Future characterization experiments would explore enrichment of opposite strand transcripts of the fad-2 operon. Lastly, there were other candidate genes to investigate by bioinformatics screen that we were unsuccessful in developing PCR primers for. We are interested in sequencing the environmental isolate strains LD1 and LD2 to refine our screen and perhaps be able to design better primers for this line of investigation.


"Sensory Element Fishing": With our preliminary pull-down result, the next step is to repeat these several times in order to confirm any interactors that we are seeing. We also wish to biotinylate more naphthenic acid-like compounds in order to create a biotinylated library of naphthenic acid molecules to further our search for sensory elements. We also want to expand our "fishing" model to search for other types of sensory elements (i.e. ChIP-SEQ).


Chassis: In terms of tools, further characterization of the HspA70/RbcS2 promoter and algal luciferase is needed. The addition of a few more selectable markers suitable for use in algae would be useful. Ultimately, we also want to finish the construction and screening of chloroplast and nuclear promoter libararies in Dunaliella tertiolecta. Now that our conjugation system is up and working, we also want to start attempting to conjugate our putative system (BBa_K640006) from E. coli and into Pseudomonas.


Prototype: The next step would be to miniaturize our circuit and create a printed circuit board. Printed circuit boards have the advantage of integrated wires, meaning the wires are small copper lines on the board instead of physical wires sticking up from the board. We will also improve the software interface that controls our potentiostat.


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