Latest revision as of 09:04, 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

In parallel of the experiments, we sent our newly designed probe to sequencing. Unfortunately, the insert was not found in the sequence when the alignment was done, the size was not the right one and the only thing that we could find was the sequence of BBa_J04450.

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

Unfortunately, results were too heterogeneous to bring any conclusions. Indeed, the number of colonies, for the 1:100 and 1:200 ratios, expected was not good enough (there is a possibility that the detector was badly digested and was consequently badly transformed).

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 36bp).
The algorithm can be found here :

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

Conclusion

In conclusion, the result of the sequencing allows us to affirm that the insertion of the probe into the backbone did not work. This explains the heterogeneous results of the sensitivity tests. Our work is based on the projects of Ireland Cork 2015 and Grenoble 2017 but the cloning technic used is different. Therefore, an optimization work has to be done to reach the expected result for construction and activation of the probes. Nonetheless, the good results obtained by these teams when using their plasmid probes suggest that the probe should be able to detect its target efficiently. We would still have to try sensibility tests to see if it would actually work, and then proceed with specificity tests to evaluate the efficiency of our probe in the context of our system, namely in a sample with the target and a lot of non-specific DNA sequences.

User Reviews

UNIQ485d91a98dcc107b-partinfo-00000000-QINU UNIQ485d91a98dcc107b-partinfo-00000001-QINU

@@ Line 2: / Line 2: @@
-<p>Experiments were done on a plasmid in which the probe has been inserted, thanks the Gibson technic, in psB1C3-BBa_J04450. </p>
+<p>Experiments were done on a plasmid in which the probe has been inserted, thanks to the Gibson method, in psB1C3-BBa_J04450. </p>
 <h1> Results of the clonning of the probe into psB1C3-BBa_J04450 </h1>
-Sequencage
+<p>In parallel of the experiments, we sent our newly designed probe to sequencing. Unfortunately, the insert was not found in the sequence when the alignment was done, the size was not the right one and the only thing that we could find was the sequence of BBa_J04450. </p>
 <h1> Test A: <I> Is this part able to detect the target for which it has been designed ?</I> </h1><br>
-mettre schema
+<center> https://static.igem.org/mediawiki/parts/0/07/T--grenoble-alpes--A3.png </center>
+<p> Unfortunately, results were too heterogeneous to bring any conclusions. Indeed, the number of colonies, for the 1:100 and 1:200 ratios, expected was not good enough (there is a possibility that the detector was badly digested and was consequently badly transformed). </p>
 <h1> Test B: <I> Is this part able to detect specifically the target for which it has been designed ? </I> </h1>
-<p> In order to evaluate the specificity of the detector part of BBa_K2629000, different “false target” sequences have been synthesized and tested with the detector.
+<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.
-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>
+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 36bp). <br>
 The algorithm can be found here : </p><br>
 <center>https://static.igem.org/mediawiki/parts/4/4b/T--grenoble-alpes--algo.png</center>
-<p>These 50%, 75%, 85%, 95% homologous sequences to the target were tested 6 times.
+<p> Unfortunately, we did not have the opportunity and the time to carry out these experiments. </p>
-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>
+<h1> Conclusion</h1>
+<b>In conclusion, the result of the sequencing allows us to affirm that the insertion of the probe into the backbone did not work. This explains the heterogeneous results of the sensitivity tests.
+Our work is based on the projects of Ireland Cork 2015 and Grenoble 2017 but the cloning technic used is different. Therefore, an optimization work has to be done to reach the expected result for construction and activation of the probes.
+Nonetheless, the good results obtained by these teams when using their plasmid probes suggest that the probe should be able to detect its target efficiently. We would still have to try sensibility tests to see if it would actually work, and then proceed with specificity tests to evaluate the efficiency of our probe in the context of our system, namely in a sample with the target and a lot of non-specific DNA sequences.</b>
 ===User Reviews===

Difference between revisions of "Part:BBa K2629000:Experience"