Difference between revisions of "Part:BBa K2629001:Experience"
 
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<p>Experiments were done on a plasmid in which the probe has been inserted, thanks the Gibson technic, in psB1C3-BBa_J04450. </p>
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<p>Experiments were done on a plasmid in which the probe has been inserted, thanks to the Gibson method, in psB1C3-BBa_J04450. </p>
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<h1> Results of the clonning of the probe into psB1C3-BBa_J04450 </h1>
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This is the alignment between psB1C3-BBa_K2629001 and the probe after probe activation by digestion.
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<center>https://static.igem.org/mediawiki/parts/9/93/T--grenoble-alpes--sequencageRESRFP.png </center>
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Unfortunately, this is not the result that we expected: the sequencing shows that the cloning did not work very well for psB1C3-BBa_K2629001. Indeed, 3 mutations can be noted and should not be present.
  
 
<h1> Test A: <I> Is this part able to detect the target for which it has been designed ?</I> </h1><br>
 
<h1> Test A: <I> Is this part able to detect the target for which it has been designed ?</I> </h1><br>
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<p>Sensitivity measures the limit of detection of our test. Here, it is defined by the lowest detectable amount of <i>Pseudomonas aeruginosa's </i> DNA that the system can detect to attests the presence of the pathogen.</p>
  
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mettre schema
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<center> https://static.igem.org/mediawiki/parts/e/eb/T--grenoble-alpes--A4.png</center>
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<p> Unfortunately, results were too heterogenous to bring any conclusions. Indeed, the number of colonies expected, for the 1:100 and 1:200 ratios, was not good enough. These results are coherent with the sequencing result. In fact, we observed that the probes were not activated despite the digestions. It is possible that the detector was not well digested and consequently not well hybridized with the target, which led to inconsistent transformation results. </p>
  
 
<h1> Test B: <I> Is this part able to detect specifically the target for which it has been designed ? </I> </h1>
 
<h1> Test B: <I> Is this part able to detect specifically the target for which it has been designed ? </I> </h1>
  
  
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<p> In order to evaluate the specificity of the detector part of BBa_K2629001, different “false target” sequences have been synthesized and tested with the detector.  
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<p> Another limitation driven by the kit is the purity of the sample. Indeed, the detection occurs when the target is mixed with a lot of foreign and unknown DNAs.
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An algorithm was made by iGEM Grenoble Alpes 2017 to give random sequences, more or less homologous to the original target with 5%, 15%, 25% and 50% randomly modified pairs of nucleotides (length is kept 36bp). <br>
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To estimate specificity, i.e. the ability of the detector to identify the true positive, the detector has to be tested with “false target sequences”, more or less homologous to the original targets. To do so, we used an algorithm made by iGEM Grenoble 2017 in order to give random sequences with 5%, 15%, 25% and 50% randomly modified pairs of nucleotides (length is kept at 42bp). In addition, the probe detects a DNA fragment with mutations causing resistance. As a result, three other controls have been added:<br>
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- One without the 2 mutations<br>
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- One with the first mutation<br>
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- One with the second mutation<br>
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  <br>
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The algorithm can be found here : </p><br>
 
The algorithm can be found here : </p><br>
  
 
<center>https://static.igem.org/mediawiki/parts/9/96/T--grenoble-alpes--RESalgo.png</center>
 
<center>https://static.igem.org/mediawiki/parts/9/96/T--grenoble-alpes--RESalgo.png</center>
  
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<p>These 50%, 75%, 85%, 95% homologous sequences to the target were tested 6 times.
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<p>Unfortunately, we did not have the opportunity and the time to carry out these experiments. </p>
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Unfortunately, results were too heterogenous to bring any conclusions, notably about the number of false positive CFU (there is a possibility that the detector was badly digested and was consequently transformed) or the supposedly increasing number of colonies. Indeed, transformation includes lot of steps with specific parameters, that all vary a little between each experiment and may explain the lack of reproducibility. </p>
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<h1>Conclusion</h1>
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<b>Unlike the probe characterizing lysis, the probe characterizing a resistance marker was inserted (not perfectly because we found 3 mutations, but we were still able to activate it). However, the activation by enzymatic digestion did not work. The main hypothesis is that the digestions did not work well due to a bad optimization of the experiment.
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This would explain the heterogeneous results of the sensitivity tests. </b>
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===User Reviews===
 
===User Reviews===

Latest revision as of 09:05, 14 October 2018


Experiments were done on a plasmid in which the probe has been inserted, thanks to the Gibson method, in psB1C3-BBa_J04450.

Results of the clonning of the probe into psB1C3-BBa_J04450

This is the alignment between psB1C3-BBa_K2629001 and the probe after probe activation by digestion.

T--grenoble-alpes--sequencageRESRFP.png

Unfortunately, this is not the result that we expected: the sequencing shows that the cloning did not work very well for psB1C3-BBa_K2629001. Indeed, 3 mutations can be noted and should not be present.

Test A: Is this part able to detect the target for which it has been designed ?


Sensitivity measures the limit of detection of our test. Here, it is defined by the lowest detectable amount of Pseudomonas aeruginosa's DNA that the system can detect to attests the presence of the pathogen.

T--grenoble-alpes--A4.png

Unfortunately, results were too heterogenous to bring any conclusions. Indeed, the number of colonies expected, for the 1:100 and 1:200 ratios, was not good enough. These results are coherent with the sequencing result. In fact, we observed that the probes were not activated despite the digestions. It is possible that the detector was not well digested and consequently not well hybridized with the target, which led to inconsistent transformation results.

Test B: Is this part able to detect specifically the target for which it has been designed ?


Another limitation driven by the kit is the purity of the sample. Indeed, the detection occurs when the target is mixed with a lot of foreign and unknown DNAs. To estimate specificity, i.e. the ability of the detector to identify the true positive, the detector has to be tested with “false target sequences”, more or less homologous to the original targets. To do so, we used an algorithm made by iGEM Grenoble 2017 in order to give random sequences with 5%, 15%, 25% and 50% randomly modified pairs of nucleotides (length is kept at 42bp). In addition, the probe detects a DNA fragment with mutations causing resistance. As a result, three other controls have been added:
- One without the 2 mutations
- One with the first mutation
- One with the second mutation

The algorithm can be found here :


T--grenoble-alpes--RESalgo.png

Unfortunately, we did not have the opportunity and the time to carry out these experiments.


Conclusion

Unlike the probe characterizing lysis, the probe characterizing a resistance marker was inserted (not perfectly because we found 3 mutations, but we were still able to activate it). However, the activation by enzymatic digestion did not work. The main hypothesis is that the digestions did not work well due to a bad optimization of the experiment. This would explain the heterogeneous results of the sensitivity tests.


User Reviews

UNIQ3257338f625d41fe-partinfo-00000000-QINU UNIQ3257338f625d41fe-partinfo-00000001-QINU