Difference between revisions of "Team:US AFRL CarrollHS/Results"

 
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       <img src="https://static.igem.org/mediawiki/2018/c/ca/T--US_AFRL_CarrollHS--ResultsHeader.jpg" alt="Results">
 
       <img src="https://static.igem.org/mediawiki/2018/c/ca/T--US_AFRL_CarrollHS--ResultsHeader.jpg" alt="Results">
 
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<img src="https://static.igem.org/mediawiki/2018/2/22/T--US_AFRL_CarrollHS--DetectCircuit.png" height="200px">
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<img src="https://static.igem.org/mediawiki/2018/8/81/T--US_AFRL_CarrollHS--RhlRPlasmid.png" style="width: 90%;">
 
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<div class="col-sm-12 label"><p class="text-center">Figure 1. Genetic Circuit for the detect and deliver mechanism</p></div>
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<div class="row"><p>With this goal in mind, the LabPats set to work with the “Detect” and “Deliver” mechanism. In their lab, Dr. Goodson had a similar construct readily available, however the gene expressed was GFP. In the first couple weeks, the LabPats were able to ligate Dr. Goodson’s construct within the iGEM backbone. This plasmid was sent off to IDT and sequenced, and the LabPats were ecstatic to see a perfectly expected sequence. They then utilized primers to insert the CheZ in place of the GFP. This proved to be quite a task as we experimented different methods to insert the CheZ within the plasmid correctly. After multiple failed attempts, the LabPats tried it over and over again, going as far as experimenting with a gradient PCR and ordering brand new primers. Eventually, after a lot of trials, the LabPats were able to send off the final construct to IDT for sequencing, achieving favorable results.</p>
 
<div class="row"><p>With this goal in mind, the LabPats set to work with the “Detect” and “Deliver” mechanism. In their lab, Dr. Goodson had a similar construct readily available, however the gene expressed was GFP. In the first couple weeks, the LabPats were able to ligate Dr. Goodson’s construct within the iGEM backbone. This plasmid was sent off to IDT and sequenced, and the LabPats were ecstatic to see a perfectly expected sequence. They then utilized primers to insert the CheZ in place of the GFP. This proved to be quite a task as we experimented different methods to insert the CheZ within the plasmid correctly. After multiple failed attempts, the LabPats tried it over and over again, going as far as experimenting with a gradient PCR and ordering brand new primers. Eventually, after a lot of trials, the LabPats were able to send off the final construct to IDT for sequencing, achieving favorable results.</p>
 
<p>While in the design phase of constructing their plasmid, the students also completed motility tests with <i>E. coli</i> cells with the CheZ knocked out. <i>E. coli</i> by nature have produce CheZ, so by testing with a strain without CheZ (courtesy of Professor Sandy Parkinson), the LabPats were able to make a clear distinction that the <i>E. coli</i> without CheZ knocked out were non-motile. Thus, they could insert their finished plasmid within the non-motile <i>E. coli</i> and observe movement towards C4-HSL.</p></div>
 
<p>While in the design phase of constructing their plasmid, the students also completed motility tests with <i>E. coli</i> cells with the CheZ knocked out. <i>E. coli</i> by nature have produce CheZ, so by testing with a strain without CheZ (courtesy of Professor Sandy Parkinson), the LabPats were able to make a clear distinction that the <i>E. coli</i> without CheZ knocked out were non-motile. Thus, they could insert their finished plasmid within the non-motile <i>E. coli</i> and observe movement towards C4-HSL.</p></div>
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<div class="row"><p>We also performed some experiments with GFP to ensure the detect mechanism worked correctly, and these results can be found on our <a href="https://2018.igem.org/Team:US_AFRL_CarrollHS/Demonstrate">Demonstrate</a> page.</p></div>
  
 
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<div class="row"><h1>Destroy</h1></div>
 
<div class="row"><h1>Destroy</h1></div>
 
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<div class="row"><p>Chitinase and Cinnamaldehyde Testing:
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Four different concentration combinations of cinnamaldehyde and chitinase were
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Chitinase C-1 was successfully cloned with the ice nucleation protein into the iGEM backbone plasmid for part submission.
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placed on top of growing Yarrowia Lipolytica, a known isolate in biodiesel
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Chitinase B4A was successfully cloned with the ice nucleation protein into the iGEM backbone plasmid for part submission.
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contamination. The goal of the experiment was to discover an effective combination of
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The three enzymes in order to produce cinnamaldehyde, which was originally in three parts, was assembled into a DNA vector with two more ribosomal binding sites to improve efficiency, but was not able to be successfully cloned into the iGEM backbone plasmid.  
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concentrations that would result in the elimination of the fungal isolate. The four
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different concentration combinations were Chitinase enzyme from Streptomyces
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<p>We also performed a number of tests to determine the efficacy of chitinase and cinnamaldehyde against fungi and bacteria, and these results can be found on our <a href="https://2018.igem.org/Team:US_AFRL_CarrollHS/Demonstrate">Demonstrate</a> page</p>
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Griseus diluted 10 mg/mL mixed with 0.66 mg/mL diluted cinnamaldehyde, Chitinase
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enzyme from Streptomyces Griseus diluted 5 mg/mL mixed with 0.66 mg/mL diluted
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cinnamaldehyde, Chitinase enzyme from Streptomyces Griseus diluted 10 mg/mL
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mixed with 0.33 mg/mL diluted cinnamaldehyde, and Chitinase enzyme from
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Streptomyces Griseus diluted 5 mg/mL mixed with 0.33mg/mL diluted cinnamaldehyde.
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After growing the plates overnight at 27 degrees Celsius for between 14 and 16 hours,
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the plates were determined to have shown the expected results. The start of a zone of
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clearing can be viewed on the plates on every concentration combination, but none
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completely eliminated all of the Yarrowia Lipolytica resulting in a complete zone of
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clearing. Further testing needs to occur before any association can be made between
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Cinnamaldehyde Biofilm Assay Testing:
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In order to test cinnamaldehyde’s preventative abilities against biofilm formation,
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multiple experiments were conducted against Nissle E. coli, a bacteria known for
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growing biofilms. The goal of the experiment was to discover the half maximal effective
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concentration (EC 50 ) of cinnamaldehyde that would be effective in preventing biofilm
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formation. The results of the experiments showed that on average, the lowest
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concentration of cinnamaldehyde required was 0.26 mg/mL to remove all growth.
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<div class="col-sm-12 label"><p class="text-center">Figure 2. Genetic Circuit for the chitinase mechanism</p></div>
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Further testing needs to be completed in order to find the range between 0 and 0.26
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mg/mL that will give the EC 50 .</p></div>
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Latest revision as of 03:58, 18 October 2018

Results

Detect and Deliver

The LabPats were able to complete their sense and respond module as planned. The goal of the project was to base a plasmid design very similar to last years. The students wanted a simple circuit design capable of delivering the microbe to the C4-HSL and biofilm. They first started with a constitutive promoter, PJ23117, to continually express RhlR. By continuing expressing RhlR, it would allow for the binding of C4-HSL whenever it became present. Afterwards, it would activate the PRhl promoter and start the transcription and translation of our CheZ gene, which would allow the flagellum of the microbe to navigate in a straight line path. When C4-HSL was no longer expressed, CheZ production would stop and the degradation tag would kick in, allowing the cell to start tumbling again. Hopefully through expressing CheZ and tumbling, a majority of the bacterium will be able to make it to the biofilm.

Figure 1. Genetic Circuit for the detect and deliver mechanism


With this goal in mind, the LabPats set to work with the “Detect” and “Deliver” mechanism. In their lab, Dr. Goodson had a similar construct readily available, however the gene expressed was GFP. In the first couple weeks, the LabPats were able to ligate Dr. Goodson’s construct within the iGEM backbone. This plasmid was sent off to IDT and sequenced, and the LabPats were ecstatic to see a perfectly expected sequence. They then utilized primers to insert the CheZ in place of the GFP. This proved to be quite a task as we experimented different methods to insert the CheZ within the plasmid correctly. After multiple failed attempts, the LabPats tried it over and over again, going as far as experimenting with a gradient PCR and ordering brand new primers. Eventually, after a lot of trials, the LabPats were able to send off the final construct to IDT for sequencing, achieving favorable results.

While in the design phase of constructing their plasmid, the students also completed motility tests with E. coli cells with the CheZ knocked out. E. coli by nature have produce CheZ, so by testing with a strain without CheZ (courtesy of Professor Sandy Parkinson), the LabPats were able to make a clear distinction that the E. coli without CheZ knocked out were non-motile. Thus, they could insert their finished plasmid within the non-motile E. coli and observe movement towards C4-HSL.

We also performed some experiments with GFP to ensure the detect mechanism worked correctly, and these results can be found on our Demonstrate page.

Destroy

Chitinase C-1 was successfully cloned with the ice nucleation protein into the iGEM backbone plasmid for part submission. Chitinase B4A was successfully cloned with the ice nucleation protein into the iGEM backbone plasmid for part submission. The three enzymes in order to produce cinnamaldehyde, which was originally in three parts, was assembled into a DNA vector with two more ribosomal binding sites to improve efficiency, but was not able to be successfully cloned into the iGEM backbone plasmid.

We also performed a number of tests to determine the efficacy of chitinase and cinnamaldehyde against fungi and bacteria, and these results can be found on our Demonstrate page

Figure 2. Genetic Circuit for the chitinase mechanism

Contact us @iGEMLabPats or iGEM@carrollhs.org