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− | We decided to order gBlocks of optimized FRB and FKBP sequence instead of optimizing the FRB and FKBP coding sequences we got from Takara Clontech. This decision was only taken for time-saving considerations. We assumed that direct mutations for optimizing the coding sequence of FRB and FKBP were | + | We decided to order gBlocks of optimized FRB* and FKBP sequence instead of optimizing the FRB and FKBP coding sequences we got from Takara Clontech ( * shows the optimization that we perform on FRB sequence to be more adaptable to bacteria system). This decision was only taken for time-saving considerations. We assumed that direct mutations for optimizing the coding sequence of FRB and FKBP and eliminating forbidden site were time-consuming steps. |
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+ | To start we inserted each gBlocks in the intermediate plasmid and then extracted the plasmid coding for our gBlocks. Then by PCR amplification we obtained our desired sequences and with the assembly techniques such as ligation and Gibson we assembled different gBlocks together. We successfully constructed the plasmid containing our Biobrick coding for FRB-GFP11 (illustrated on '''[fig. 1]''') and FKBP-GFP10 (illustrated on '''[fig. 2]'''). | ||
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[[File: T--Paris Saclay--project result Fig1.jpg|500px|centre|]] | [[File: T--Paris Saclay--project result Fig1.jpg|500px|centre|]] | ||
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[[File:T--Paris Saclay--project result Fig3.jpg|650px|centre|]] | [[File:T--Paris Saclay--project result Fig3.jpg|650px|centre|]] | ||
<center><b>Figure 3:</b> Map of the plasmid pSB1C3 coding for FKBP-GFP10 and FRB-GFP11</center> | <center><b>Figure 3:</b> Map of the plasmid pSB1C3 coding for FKBP-GFP10 and FRB-GFP11</center> | ||
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+ | ===Characterization=== | ||
+ | To characterize our system, we tested the expression and integrity of the three protein compounds of the system with western blot. Unfortunately, we could not obtain polyclonal anti-GFP and we used the monoclonal one. As we were afraid the monoclonal antibody recognized only one subunit of the tripartite GFP which was GFP1.9. Here we are certain that GFP1.9 expressed correctly. However, in the case of GFP10 and GFP11, our negative results of western blot are not informative because we are not certain if there are not protein expressed or it did not detect. Here we showed that the monoclonal anti-GFP antibody epitope are on GFP1.9. | ||
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[[File: T--Paris Saclay--WesternGFP.png|500px|centre|]] | [[File: T--Paris Saclay--WesternGFP.png|500px|centre|]] | ||
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On this western blot control GFP appears between 28 and 38 kDa. GFP1.9 subunit is also revealed with a lower weight as expected. But GFP10 and GFP11 are negative. | On this western blot control GFP appears between 28 and 38 kDa. GFP1.9 subunit is also revealed with a lower weight as expected. But GFP10 and GFP11 are negative. | ||
</center> | </center> | ||
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+ | ===Test of the dimerization system and tripartite GFP=== | ||
+ | We transformed ''E.coli'' with pSB1C3 coding for FKBP-GFP10 and FRB-GFP11 and pUC19 coding for the third part of GFP as we could not clone GFP1.9 in pSB1C3. Transformed cells where then incubated with the dimerization agent, rapalog. We try several concentrations of rapalog but we did not see any GFP fluorescence with flow cytometer. | ||
+ | [[File: T--Paris Saclay--Testbiobrick1.png|600px|centre|]] | ||
+ | <center><b>Figure 5:</b> : No GFP florescence is observe with rapalog | ||
+ | <br>All the rapalog concentrations tested (5nM, 50nM, 500nM) display a signal inferior compared to the negative control. </center> | ||
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+ | We did not obtain the fluorescent signal that we expected with the dimerization agent. We did not have time to investigate further why the system did not work, but we have several ideas. We may have some expression issues with FRB*-GFP11 and FKBP-GFP10 as the western blot done with monoclonal antibody anti-GFP allow the revelation of GFP1.9 only. Another possibility is that there is some physical constraint that prevent GFP assembly, the linker length for example can generate this kind of constraint. Finally, the system can by well-constructed and expressed but we may not have found the optimal conditions to induce the dimerization (concentration of rapalog, timing, temperature). | ||
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===Visualization tool characterization=== | ===Visualization tool characterization=== | ||
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We successfully inserted all the gBlocks into intermediate plasmids. However, due to a lack of time, we could not achieve the final construction of our plasmid (pZA11) by Gibson assembly method. | We successfully inserted all the gBlocks into intermediate plasmids. However, due to a lack of time, we could not achieve the final construction of our plasmid (pZA11) by Gibson assembly method. | ||
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==Construction of the two different systems== | ==Construction of the two different systems== | ||
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===Bring DNA Closer (BDC) tool construction=== | ===Bring DNA Closer (BDC) tool construction=== | ||
− | The BDC tool construct is composed of the dCas9 | + | The BDC tool construct is composed of the dCas9 ''S. p'' linked to FRB* and the dCas9 ''T. d'' linked to FKBP12 (for further details, check our [https://2016.igem.org/Team:Paris_Saclay/Design Design page]. To build this tool, [http://www.addgene.org/48657/ dCas9 ''S. p''] and [http://www.addgene.org/48660/ dCas9 ''T. d'']) were ordered from [https://www.addgene.org/ addgene.org], and the genes encoding the optimized FRB and FKBP12 were obtained by ordering gBlocks from [http://eu.idtdna.com/site IDT] '''[Table 1]'''. |
As plasmids we received from [https://www.addgene.org/ addgene.org]were already transformed into the bacteria, we prepared the overnight culture of received bacteria in order to extract the plasmid coding for dCas9. In this part of the project, we faced some difficulties because the plasmids were low copy number. We used a midi-prep kit in order to extract enough plasmids. However, according to the results of PCRs on the extracted plasmids, we could not amplify the coding gene of the dCas9s. Furthermore, we sent plasmids for sequencing. We found out that the 3’ end of the gene encoding dCas9 Td did not match the expected sequence. | As plasmids we received from [https://www.addgene.org/ addgene.org]were already transformed into the bacteria, we prepared the overnight culture of received bacteria in order to extract the plasmid coding for dCas9. In this part of the project, we faced some difficulties because the plasmids were low copy number. We used a midi-prep kit in order to extract enough plasmids. However, according to the results of PCRs on the extracted plasmids, we could not amplify the coding gene of the dCas9s. Furthermore, we sent plasmids for sequencing. We found out that the 3’ end of the gene encoding dCas9 Td did not match the expected sequence. | ||
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<div style="width:65%; float:right;"> | <div style="width:65%; float:right;"> | ||
[[File:T--Paris_Saclay--project_2016_dcas.jpeg|500px|center|]] | [[File:T--Paris_Saclay--project_2016_dcas.jpeg|500px|center|]] | ||
− | <center><b>Figure | + | <center><b>Figure 6:</b> Scheme of the visualization tool design. Visualization tool is composed of three main parts: the biobrick containing dCas9 NM linked to GFP10 coding sequences, the biobrick containing dCas9 ST linked to GFP11 coding sequences, and the biobrick containing the GFP 1-9 coding sequence. The two first biobricks are split into 2 fragments, themselves split into 2 gBlocks.</center> |
</div> | </div> | ||
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The first step was to check gBlocks, because according to IDT, 80% of the gBlocks have the correct ordered sequence. In order to check them, gBlocks were cloned into pUC19 in first attempt, or pJET plasmid for difficult cases. pUC19 is a plasmid that allows white/blue screening, and pJET is a plasmid that allows only cells containing the plasmid cloned with an insert to grow on selective media. Positive colonies were screened by colony PCR, using the universal pUC19 or pJET vectors. PCR products were checked using gel electrophoresis. '''[Fig. 5]''' shows the results for gBlock 3.1. In this particular case, the expected fragment was 960bp. Results showed that clones 2 and 5 were positive. Plasmid DNA selected by PCR colony screening were cultivated then extracted to be sent for sequencing. | The first step was to check gBlocks, because according to IDT, 80% of the gBlocks have the correct ordered sequence. In order to check them, gBlocks were cloned into pUC19 in first attempt, or pJET plasmid for difficult cases. pUC19 is a plasmid that allows white/blue screening, and pJET is a plasmid that allows only cells containing the plasmid cloned with an insert to grow on selective media. Positive colonies were screened by colony PCR, using the universal pUC19 or pJET vectors. PCR products were checked using gel electrophoresis. '''[Fig. 5]''' shows the results for gBlock 3.1. In this particular case, the expected fragment was 960bp. Results showed that clones 2 and 5 were positive. Plasmid DNA selected by PCR colony screening were cultivated then extracted to be sent for sequencing. | ||
[[File: T--Paris Saclay--project 2016 2.jpg|300px|centre|]] | [[File: T--Paris Saclay--project 2016 2.jpg|300px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 7:</b> Colony PCR products gel electrophoresis of gBlock 3.1. Expected fragment: 960bp. Clones 2 and 5 are positive.</center> |
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When sequences were as expected, the same plasmidic DNA extraction products were used to perform high fidelity PCRs, using primers which hybridized at both extremities of the gBlock. These PCR products were then stocked or used to perform ligations, and the ligation products were amplified by high fidelity PCRs. '''[Fig. 6]''' shows the electrophoresis gel of PCR amplifications of ligations made in order to obtain fragments 3 and 4. The expected fragments size are respectively 1920bp and 1994 bp. | When sequences were as expected, the same plasmidic DNA extraction products were used to perform high fidelity PCRs, using primers which hybridized at both extremities of the gBlock. These PCR products were then stocked or used to perform ligations, and the ligation products were amplified by high fidelity PCRs. '''[Fig. 6]''' shows the electrophoresis gel of PCR amplifications of ligations made in order to obtain fragments 3 and 4. The expected fragments size are respectively 1920bp and 1994 bp. | ||
[[File: T--Paris Saclay--project 2016 3.png |400px|centre|]] | [[File: T--Paris Saclay--project 2016 3.png |400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 8:</b> Gel electrophoresis of ligation products amplified by high fidelity PCR for fragments 3 and 4. Expected size were respectively 1920 and 1994 bp. Results showed that fragments with the correct size were obtained.</center> |
Thanks to an overlap between the two fragments (1 and 2, 3 and 4), an overlap between the prefix present at the beginning of fragment 1 and 3 and pSB1C3, and an overlap between the suffix present at the end of fragment 2 and 4 and pSB1C3, we were able to assemble them in pSB1C3 using the Gibson Assembly method. Gibson Assembly products are then transformed into bacteria, and a colony PCR was performed using primers complementary to the suffix and prefix sequences to screen positive bacteria colonies. PCR products are then checked on gel electrophoresis. '''[Fig. 7]''' shows the results of this screening for the assembly of fragments 3 and 4 in pSB1C3. The expected band size is approximately 4000 bp. This fragment was obtained for colonies 6 and 8. | Thanks to an overlap between the two fragments (1 and 2, 3 and 4), an overlap between the prefix present at the beginning of fragment 1 and 3 and pSB1C3, and an overlap between the suffix present at the end of fragment 2 and 4 and pSB1C3, we were able to assemble them in pSB1C3 using the Gibson Assembly method. Gibson Assembly products are then transformed into bacteria, and a colony PCR was performed using primers complementary to the suffix and prefix sequences to screen positive bacteria colonies. PCR products are then checked on gel electrophoresis. '''[Fig. 7]''' shows the results of this screening for the assembly of fragments 3 and 4 in pSB1C3. The expected band size is approximately 4000 bp. This fragment was obtained for colonies 6 and 8. | ||
[[File: T--Paris Saclay--project 2016 4.png |400pxcentre|]] | [[File: T--Paris Saclay--project 2016 4.png |400pxcentre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 9:</b> Migration of colony PCR products obtained from bacteria transformed with the Gibson Assembly of Fragments 3 and 4 in pSB1C3. The expected product was around 4000 bp and it was found at the expected size for clones 6 and 8.</center> |
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[[File:T--Paris_Saclay--project_2014_1.jpeg.png|400px|centre|]] | [[File:T--Paris_Saclay--project_2014_1.jpeg.png|400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 10:</b> Schema of the lemon ripening project. The decrease of salicylate concentration causes a lost of suppressor tRNA and so on the fall of blue chromoprotein expression : bacteria changes from green to yellow.</center> |
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[[File:T--Paris_Saclay--project_2014_2.jpeg|400px|centre|]] | [[File:T--Paris_Saclay--project_2014_2.jpeg|400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 11:</b> Explanatory diagram of the lemon ripening. NahR becomes active in presence of salicylate : there is expression of suppressor tRNA. This one suppresses amber codon allowing the translation of the green fusion chromoprotein.</center> |
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[[File:T--Paris Saclay--20161015 characterizationk1372001.png|400px|centre|]] | [[File:T--Paris Saclay--20161015 characterizationk1372001.png|400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 12:</b> Explanatory diagram of the characterization. pcl_TAA construction contains a TAA stop codon between LacZ and Luc. pcl_Tq construction does not contain any stop codon. pcl_TAG contains the TAG codon recognized by supD suppressor t-RNA.</center> |
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[[File:T--Paris_Saclay--activity_Luc_Gal_Tq_fonction_salicylate2.PNG|400px|centre|]] | [[File:T--Paris_Saclay--activity_Luc_Gal_Tq_fonction_salicylate2.PNG|400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 13:</b> Luciferase activity with TQ construction depending of salicylate concentration. 3 clones were tested per condition. Luciferase activity depends on the transcription of pclTAA.</center> |
The Tq plasmid does not contain any stop codon between LacZ and Luc. Thus, no matter the salicylate concentration, both Luciferase and Beta Galactosidase activities are supposed to be detected. | The Tq plasmid does not contain any stop codon between LacZ and Luc. Thus, no matter the salicylate concentration, both Luciferase and Beta Galactosidase activities are supposed to be detected. | ||
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[[File:T--Paris_Saclay--activity_Luc_Gal_TAA_fonction_salicylate2.PNG|400px|centre|]] | [[File:T--Paris_Saclay--activity_Luc_Gal_TAA_fonction_salicylate2.PNG|400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 14:</b> Luciferase activity with TAA construction depending of salicylate concentration. 3 clones were tested per condition. Luciferase activity depends on the expression and capacity of the suppressor t-RNA to read TAA codon.</center> |
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[[File:T--Paris_Saclay--activity_Luc_Gal_TAG_fonction_salicylate2.PNG|400px|centre|]] | [[File:T--Paris_Saclay--activity_Luc_Gal_TAG_fonction_salicylate2.PNG|400px|centre|]] | ||
− | <center><b>Figure | + | <center><b>Figure 15:</b> Luciferase activity with TAG construction depending of salicylate concentration. 3 clones were tested per condition. Luciferase activity depends on the expression and capacity of the suppressor t-RNA to read TAG codon.</center> |
Latest revision as of 00:07, 20 October 2016