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 cloning of the BFP gene instead of RFP gene and the probe cloning

There are the alignments realized:

- between psB1C3-BBa_K2629003 and new BFP gene (A).
- between psB1C3-BBa_K2629003 and the probe after probe activation by PCR linearization (B).

Unfortunately, this is not the result that we expected. In fact, the sequencing shows that the cloning of the probe did not work. Regrettably, we did not have the time to make more sensibility tests. However, the substitution of RFP gene by BFP gene did work well as we can see one the figure A. This means that a new biobrick was constructed with the same RBS, promoter and terminator system as BBa_J04450 but with another gene than mRFP E1010 --> K592100 (BFP gene).

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 expected, for the 1:100 and 1:200 ratios, 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 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 :

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

Conclusion

In conclusion, this probe aims to characterize a resistance marker of Pseudomonas aeruginosa. If the constructed probe recognizes the target, it hybridizes, the plasmid is circular and ready to be transformed. After this transformation in TOP10, the bacteria express the reporter gene which is blue in this case. The idea of our project is to associate a color with a probe. The red color (RFP) for the probe characterizing the lysis of Pseudomonas aeruginosa and the blue color for the probe characterizing a resistance marker of Pseudomonas aeruginosa.

Finally, the probe was not inserted in this case. On the other hand, the substitution of the gene of the RFP by the gene of the BFP was a success. Unfortunately, we did not have time to properly characterize this new biobrick. However, it could be used in several applications. As we have tried, it can be used in diagnosis with a report color.

User Reviews

UNIQ4b9ffed593d38555-partinfo-00000000-QINU UNIQ4b9ffed593d38555-partinfo-00000001-QINU

@@ Line 1: / Line 1: @@
-<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 cloning of the BFP gene instead of RFP gene and the probe cloning </h1>
+There are the alignments realized: <br>
+- between psB1C3-BBa_K2629003 and new BFP gene (A). <br>
+- between psB1C3-BBa_K2629003 and the probe after probe activation by PCR linearization (B).  <br>
+<center>https://static.igem.org/mediawiki/parts/d/dc/T--grenoble-alpes--sequencageRESBFP.png</center>
+Unfortunately, this is not the result that we expected. In fact, the sequencing shows that the cloning of the probe did not work. Regrettably, we did not have the time to make more sensibility tests.
+However, the substitution of RFP gene by BFP gene did work well as we can see one the figure A. This means that a new biobrick was constructed with the same RBS, promoter and terminator system as BBa_J04450 but with another gene than mRFP E1010 -->  K592100 (BFP gene).
 <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/e/eb/T--grenoble-alpes--A4.png </center>
+<p> Unfortunately, results were too heterogeneous to bring any conclusions. Indeed, the number of colonies expected, for the 1:100 and 1:200 ratios, 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_K2629003, 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 42bp). In addition, the probe detects a DNA fragment with mutations causing resistance. As a result, three other controls have been added:<br>
+- One without the 2 mutations<br>
+- One with the first mutation<br>
+- One with the second mutation<br>
+  <br>
 The algorithm can be found here : </p><br>
 <center>https://static.igem.org/mediawiki/parts/9/96/T--grenoble-alpes--RESalgo.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, this probe aims to characterize a resistance marker of <i>Pseudomonas aeruginosa</i>. If the constructed probe recognizes the target, it hybridizes, the plasmid is circular and ready to be transformed. After this transformation in TOP10, the bacteria express the reporter gene which is blue in this case.
+The idea of our project is to associate a color with a probe.
+The red color (RFP) for the probe characterizing the lysis of <i>Pseudomonas aeruginosa</i> and the blue color for the probe characterizing a resistance marker of <i>Pseudomonas aeruginosa</i>.<br>
+Finally, the probe was not inserted in this case. On the other hand, the substitution of the gene of the RFP by the gene of the BFP was a success. Unfortunately, we did not have time to properly characterize this new biobrick. However, it could be used in several applications. As we have tried, it can be used in diagnosis with a report color.</b>
 ===User Reviews===

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