EPF-Lausanne/9 September 2009

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Wet Lab

Culture

Made one new bottle of M9/min+AA+thiamine in 500 ml.

All clones (M9 & LB) have grown. M9 cultures have been redone in 25 ml M9 + antibiotics. 750 ul of overnight re-inoculated. Put in incubator at 37°C for 2 hours. We got different OD after this time, for RBS, LacI-RFP #1,2, RO2 #4,5,10, RO1 # 1,2,3. We normalized the OD to 0.06 for all of them by adding the appropriate amount of fresh medium to each erlen' (calculation was done linear).

Characterization

With Atc

Without Atc

With Trp

1/2 Trp + 1/2 Atc 1 Trp + 1/2 Atc	1/2 Trp + 1 Atc 1 Trp + 1 Atc

1/2 Trp
1 Trp
3/2 Trp

Without Trp

1/2 Atc
1 Atc
3/2 Atc

Without Atc nor Trp

50 ul of culture in each well.

Trp : 0.5 -> 1ul, 1 -> 2ul, 1.5 -> 3 ul.

ATC : 0.5 -> 0.5 ul (so about 50 ng/ml), 1 -> 1ul.

IPTG : 1x -> 5.5 ul -> 1 mM final.

Note : from the start of the induction to putting the plate in the machine, it took about 20 minutes (so some wells may already have been induced).

Results of the characterization

ALL OF THIS HAS TO BE ANALYZED

For the RBS :

RBS was our negative control. We can see that there is no RFP fluorescence (because of course RBS has no RFP gene).

For LacI-RFP 1 + IPTG :

LacI-RFP 2 + IPTG show the same tendancy.

For Read Out 1

RO1#1 without TRP:

Without any tryptophan, the system should be on : TrpO isn't repressed so RFP is expressed. Here we can see that without TRP the cells express some RFP fluorescence.

Clone #2 has the same behaviour :

RO1#1 + 0.5 TRP :

In comparison to RO1#1 without TRP, if we add some TRP, there is a significative difference. TrpO is inhibited and RFP isn't expressed any more. We can see here that our system seems to work, so RO1 is characterized.

RO1#2 + 0.5 TRP shows the same tendancy :

RO1#1 + 1 TRP :

Again, RO1#2 clone has the same behaviour :

RO1#3 clones doesn't follow these tendancy. It actually seems that this clone isn't working at all. It is possible a spontaneous mutation occured in some place... On all different conditions, RO1#3 clones show a flat curve, like RBS. It doesn't express RFP which means either our construct wasn't inserted, either there has been a mutation in it. For example + 1 TRP :

For Read Out 2

RO2#1 without TRP :

RO2#3 has exactly the same curve shape :

RO2#1 + 0.5 TRP :

RO2#3 has a more "normal" shape :

RO2#1 + 1 TRP :

RO2#3 is the same :