EPF-Lausanne/8 September 2009

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(Wet Lab)
(Wet Lab)
 
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As RO1 grew in both media, we put it in 25mL medium with Amp. We did also with RO2 (LB), and RO2+BB (LB), and LacI-RFP.
As RO1 grew in both media, we put it in 25mL medium with Amp. We did also with RO2 (LB), and RO2+BB (LB), and LacI-RFP.
-
For the characterisation, we did different combinations : -TRP, -TRP -ATC, +TRP -ATC, +TRP +ATC. Evertime for all the clones in M9 and then in LB.
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For the characterisation, we did different combinations : -TRP, -TRP -ATC, +TRP -ATC, +TRP +ATC. Every time for all the clones in M9 and then in LB.
TRP : LB : dilution 50x so 1ul, M9 : dilution 25x so 2 ul. We took the stock solution 4,250 g/L.
TRP : LB : dilution 50x so 1ul, M9 : dilution 25x so 2 ul. We took the stock solution 4,250 g/L.
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ATC : 5x dilution so 10 ul from the 500 ng/ml stock solution.
ATC : 5x dilution so 10 ul from the 500 ng/ml stock solution.
 +
The aim was to get used to the machine and to see the differences between LB or M9.
 +
 +
Here are the results of the characterization :
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 +
RO1#1 + TRP in LB
 +
There is already some TRP in LB so it shouldn't change a lot.
 +
[[Image:RO11TRPLB.jpg|center|RO1#1 + TRP in LB]]
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 +
<br>
 +
RO1#1 without TRP in LB
 +
This doesn't mean a lot as there is already some TRP in LB but we can try to see a difference with RO1#1 + TRP in LB.
 +
[[Image:RO11LB.jpg|center|RO1#1 in LB]]
 +
 +
<br>
 +
RO1#2 + TRP in LB
 +
[[Image:RO12TRPLB.jpg|center|RO1#2 + TRP in LB]]
 +
 +
<br>
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RO1#2 without TRP in LB
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[[Image:RO12LB.jpg|center|RO1#2 in LB]]
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 +
<br>
 +
RO1#3 + TRP in LB
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The clone 3 doesn't seem to be functional.
 +
[[Image:RO13TRPLB.jpg|center|RO1#3 + TRP in LB]]
 +
 +
<br>
 +
RO1#3 without TRP in LB
 +
[[Image:RO13LB.jpg|center|RO1#3 in LB]]
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 +
<br>
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RO1#3 + TRP in M9
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[[Image:RO13TRPM9.jpg|center|RO1#3 + TRP in M9]]
 +
 +
<br>
 +
RO1#3 without TRP in M9
 +
[[Image:RO13M9.jpg|center|RO1#3 in M9]]
 +
 +
<br>
 +
RO2 + TRP + ATC
 +
[[Image:RO2TRPATC.jpg|center|RO2 + TRP + ATC]]
 +
 +
<br>
 +
RO2 + TRP - ATC
 +
[[Image:RO2TRP.jpg|center|RO2 + TRP]]
 +
 +
<br>
 +
RO2 without TRP + ATC
 +
[[Image:RO2ATC.jpg|center|RO2 + ATC]]
 +
 +
<br>
 +
RO2 without TRP and without ATC
 +
[[Image:RO2without.jpg|center|RO2]]
===Results of the transformation===
===Results of the transformation===
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LB : RO2 + BB (1,3,4,8), LacI-RFP (#1,2), BB/Chl (results of the gel : 4 clones).
LB : RO2 + BB (1,3,4,8), LacI-RFP (#1,2), BB/Chl (results of the gel : 4 clones).
M9 : LacI-RFP (2 clones), RBS BBa_B0030 (control for the fluo. of the cells), RO2 (4,5,10), RO1 (1,2,3).
M9 : LacI-RFP (2 clones), RBS BBa_B0030 (control for the fluo. of the cells), RO2 (4,5,10), RO1 (1,2,3).
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===Reculture===
 
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Made one new bottle of M9 min + AA + thiamine in 500 ml.
 
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All clones (M9 & LB) have grown. M9 cultures have been re-done in 25 ml M9 + antibiotic. 750 ul of overnight re-inocculated.
 
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Put in incubator at 37°C for 2 hours.
 
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We got different OD after this time.
 
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RBS, LacI-RFP #1,2, RO2 #4,5,10, RO1 #1,2,3.
 
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We normalized the OD to 0.06 for all of them by adding the appropriate amount of fresh medium to each erlen (the calculation was done linear).
 
==People in the lab==
==People in the lab==

Latest revision as of 16:51, 20 October 2009

Contents

8 September 2009





Wet Lab

Cultures

Results:

RO1 has grown in LB and M9
RO2 has grown in LB but not in M9
RO2+BB #1,4 did not grow
RO2+BB #3 has grown in LB but not in M9

-> problem with our glycerol stock (RO2 + BB didn't grow in LB !!). We did some plates of the cultures that grew.

For safety we did some plates with the glycerol stocks of RO2 + BB 1,4,8. We also did some plates with the cultures that grew (RO1 1,2,3, RO2 4,5,10, RO2 + BB 3).


Characterization

As RO1 grew in both media, we put it in 25mL medium with Amp. We did also with RO2 (LB), and RO2+BB (LB), and LacI-RFP.

For the characterisation, we did different combinations : -TRP, -TRP -ATC, +TRP -ATC, +TRP +ATC. Every time for all the clones in M9 and then in LB.

TRP : LB : dilution 50x so 1ul, M9 : dilution 25x so 2 ul. We took the stock solution 4,250 g/L.

ATC : 5x dilution so 10 ul from the 500 ng/ml stock solution.

The aim was to get used to the machine and to see the differences between LB or M9.

Here are the results of the characterization :

RO1#1 + TRP in LB There is already some TRP in LB so it shouldn't change a lot.

RO1#1 + TRP in LB


RO1#1 without TRP in LB This doesn't mean a lot as there is already some TRP in LB but we can try to see a difference with RO1#1 + TRP in LB.

RO1#1 in LB


RO1#2 + TRP in LB

RO1#2 + TRP in LB


RO1#2 without TRP in LB

RO1#2 in LB


RO1#3 + TRP in LB The clone 3 doesn't seem to be functional.

RO1#3 + TRP in LB


RO1#3 without TRP in LB

RO1#3 in LB


RO1#3 + TRP in M9

RO1#3 + TRP in M9


RO1#3 without TRP in M9

RO1#3 in M9


RO2 + TRP + ATC

RO2 + TRP + ATC


RO2 + TRP - ATC

RO2 + TRP


RO2 without TRP + ATC

RO2 + ATC


RO2 without TRP and without ATC

RO2

Results of the transformation

They all grew : RO1#2 + BB5, RO1#1 + BB1, RO1#3 + BB3.

So we did a colony PCR to be sure that we have the double transformation, using the Taq platinium protocole.

We picked 8 clones of RO1#2 + BB5, 8 clones of RO1#1 + BB1 and 5 clones of RO1#3 + BB3.

We ran a gel to check if it has the two plasmids : for the clones that worked, we should see two bands. We ran a gel : we should get two fragments. We can see that all our clones worked (had the two constructs), except one : clone 5 for RO1#3 + BB3.

Transformation

Then we did a transformation of the ligation products : BB1, BB3, BB5, BB6 in a chlorophenical receptor, on Chl plates.

Culturing

LB : RO2 + BB (1,3,4,8), LacI-RFP (#1,2), BB/Chl (results of the gel : 4 clones). M9 : LacI-RFP (2 clones), RBS BBa_B0030 (control for the fluo. of the cells), RO2 (4,5,10), RO1 (1,2,3).

People in the lab

Mélanie, Caroline, Basile, Nicolas