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EPF-Lausanne/9 September 2009
From 2009.igem.org
Contents |
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 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.
The aim of this experiment was to see the maximal level of RFP that could be expressed. For that, we put some IPTG to induce the LacI-RFP gene. But it doesn't look like what we would have expected, the level of RFP is really low.
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 :
This clone seems to work as well if we compare this graph to the one with TRP. We have the same results as for RO1#1.
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 :
RO2#1 + 1.5 TRP :
RO2#3 in the same conditions :
RO2#1 + 0.5 TRP + 0.5 ATC :
RO2#3 in the same conditions :
RO2#1 + 0.5 TRP + 1 ATC :
RO2#3 in the same conditions :
RO2#1 + 1 TRP + 0.5 ATC :
RO2#3 in the same conditions :
RO2#1 + 1 TRP + 1 ATC :
RO2#3 in the same conditions :
RO2#1 + 0.5 ATC :
RO2#3 in the same conditions :
RO2#1 + 1 ATC :
RO2#3 in the same conditions :
RO2#1 + 1.5 ATC :
RO2#3 in the same conditions :
Clone RO2#2 is not working, it has always a flat curve like this (for ex +0.5 ATC):
Glycerol stsock
We were afraid some old glycerol stocks were destroyed, so we did some new.
Strains
We received some TRP KO strains. We will grow them overnight. We grew them in 500 ml erlen and add of 75 ml of LB. 3 strains : JRG1046 5992 (no antibiotic), JRG465 4456 (no antibiotic), JW4356-2 11110 (it is supposed to be Kana resistance but we are not sure so we did part in no antibiotic and part in 450 ul Kana).
Microscope experiment
... to be completed !! p. 114 lab book 2
Cultures
-> in M9 (3ml + antibiotics). We did the following cultures : RO1#1,2, RO2#4,5,10 and some double constructs : RO2+BB#3, RO2+BB#8, RO1#2+BB5#3, RO1#1 +BB1#1, RO1#3 + BB3#3.
People in the lab
Mélanie, Caroline, Basile, Nicolas
