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Team:Lyon-INSA-ENS/Realisation/Week9
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<h1 style="color: white;"> Week 9 </h1> | <h1 style="color: white;"> Week 9 </h1> | ||
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<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> | <FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/> | ||
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| - | <b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16 (3µL)<br/> | + | <b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16. (3µL)<br/> |
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<FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/> | <FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/> | ||
| - | Start of <b>24 well plate</b> adherence test in M63G medium with PHL818 ( positive control ), NM522 ( negative control ), S18 + Amp + CoCl2 ( in increasing concentration ) <br/> <br/><br/> | + | Start of <b>24 well plate</b> adherence test in M63G medium with PHL818<br/>( positive control ), NM522( negative control ), S18 + Amp + CoCl2<br/>( in increasing concentration ) <br/> <br/><br/><br/> |
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The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water ). | The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water ). | ||
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| + | <FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br> | ||
| + | Launched an overnight PCR using the synthetised rcn-CsgBAEFG part as the template, and primers used during the first try to obtain the csg construct : 3' primer for csgBA and 5' primer for csgEFG<br> | ||
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LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522<br/> | LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522<br/> | ||
LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/> | LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/> | ||
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| + | <br/><br/> | ||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br> | ||
| + | Revealing of the PCR : it is positive, but the negative control is positive too. PCR re-done.<br> | ||
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Start of tubes for flocculation : the same as plates (2) and (3).<br> | Start of tubes for flocculation : the same as plates (2) and (3).<br> | ||
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| + | <br/><br/> | ||
| + | <p style=" line-height : 1.5em"> | ||
| + | <FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br> | ||
| + | Revealing PCR results, they show a ~2800 bp DNA band, in accordance with the expected size.<br> | ||
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| + | </p> | ||
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| + | <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week8"/><font color="grey"><b>Previous Week</b></font></a> | ||
| + | <a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week10"/><font color="grey"><b>Next Week</b></font></a> | ||
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Latest revision as of 23:35, 21 September 2011
Week 9
From Monday the 15th of August to Friday the 19th of August 2011
Monday
Transformations and controls for future tests
Transformation and plating of NM522 with : pIG3 (3µL), pIG16. (3µL)
Tuesday
Transformations and controls for future tests
Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium.
Incubation at 37°C overnight.
Adherence Test
Start of 5mL cultures for adherence tests from the collection :
LB : NM522
LB/2 : NM522, S3, S4, S15, S19
M63 : NM522, S15, S3, S19, S4, S18
Wednesday
Transformations and controls for future tests
Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.
Start of 5mL cultures of the individual clones plated on the previous day.
Transformation and plating of NM522 on LB+Amp with : pIG6 (3µL), pIG7 (3µL), pIG24 (2µL), p56 (2µL), p10 (2µL), pIG16+p157 (3µL each), pIG16+p115(3µL each), pIG16+p127(3µL each), pIG16+p116 ( 3µL and 5µL respectively ), pIG25 (1µL).
Transformation and plating of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.
Extraction of pIG16 from S19 with the QIAGen miniprep protocol.
Adherence Test
Start of 24 well plate adherence test in M63G medium with PHL818
( positive control ), NM522( negative control ), S18 + Amp + CoCl2
( in increasing concentration )
Others
Test of antibiotics : start of 5mL cultures ( 5mL LB, 50µL antibiotic, 10µL saturated NM522) of NM522 with antibiotics to test if the antibiotic is still good for Kan, Tet, Cm and Amp
The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water ).
Re-synthetising csg-BAEFG with standard iGEM restriction sites
Launched an overnight PCR using the synthetised rcn-CsgBAEFG part as the template, and primers used during the first try to obtain the csg construct : 3' primer for csgBA and 5' primer for csgEFG
Thursday
Transformations and controls for future tests
The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.
Digestion of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.
Digestion of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive.
Adherence Test Preparation
Start of 5mL cultures for 24 well plates :
M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)
LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522
LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522
Re-synthetising csg-BAEFG with standard iGEM restriction sites
Revealing of the PCR : it is positive, but the negative control is positive too. PCR re-done.
Friday
Transformations and controls for future tests
Plating of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 (who have a double resistance) on their second resistance ( Kan ).
Extraction (QIAgen) and digestion(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.
The NM522/pIG24 (Cm) strain grows on Amp plates and not Cm. Moreover, pIG24 is a linear plasmid and should not have transformed : plating of NM522, NM522/pIG24 on LB+Amp and start of 5mL cultures ( LB+Amp ) of the same strains to test Amp efficiency or contamination of NM522 with an AmpR strain.
Adherence and Flocculation Test
Start of 24 well plates :
(1) M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration)-> to characterize rcn-csgBAEFG
(2) M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak
(3) LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak
Start of tubes for flocculation : the same as plates (2) and (3).
Re-synthetising csg-BAEFG with standard iGEM restriction sites
Revealing PCR results, they show a ~2800 bp DNA band, in accordance with the expected size.
