Paris/Results

From 2007.igem.org

(Difference between revisions)
(Experimental results)
(DGAT cloning and tryglicerid synthesis in E. Coli)
Line 44: Line 44:
=== pBAD characterisation ===
=== pBAD characterisation ===
-
=== DGAT cloning and tryglicerid synthesis in E. Coli ===
+
=== DGAT cloning and triglycerid synthesis in E. Coli ===
== Modeling results ==
== Modeling results ==

Revision as of 12:18, 21 October 2007


Contents

Experimental results

Survival of dapA deletant

If we want our system to be robust, the germ line cells should be able to live as long as possible when deprived of DAP. They may indeed face such a feat within our system if their is not enough somatic cells at a given time.

Our dapA deletant strain was grown over night in LB + 300µM DAP. A culture was then launched diluting 100 time the ON culture in LB (+ Kanamycin). 100µL were plated each hour and the number of clone determined.

dapA deletant growth

We expect the somatic cells to excrete only a limited amount of DAP, it would thus be nice if our auxotroph strain could grow on limited amount of metabolite. Over night culture were diluted 100 time in LB suplemented with different amount of DAP.

DAP excretion by prototroph cells

We want to know if prototroph cells are excreting DAP and in what amount. Instead of doing proper chemical dosages which are expensive, we had the idea to make a biological measurments.

Prototroph MG1655 strain was grown in LB, and the medium was recovered at different stages of the culture and purified. The dapA deletant strain was then grown in the recovered medium. The idea is that comparing the growth curve of this experiment with growth curves of dapA deletant in differently supplemented LB medium, should alow us to determine approimatively the amount of DAP present in the recovered medium.

Co-culture experiments

We want to know if the soma of our synthetic organism will be able to feed the germ line. Unfortunatly, this cannot properly be done without the final construct. Nevertheless, we can already check if in similar situations, dapA deleted strain can survive.

Two different co-culture experiments were performed:

  • In the first one, the dapA deleted strain is grown with a prototroph strain. In this case, we know for sure that the prototroph cells will take the population over. Nervertheless it is interesting to see if dapA deleted strain survive longer in this condition than if alone and without DAP.
  • In the second one, the dapA deleted strain is grown with a strain bearing an auxotrophy to another metabolite. This experiment reproduces cross-feeding works done on the yeast by (look for the exact reference). In this coculture there is a mutual dependence of each strain for the other. If this works for the dapA deleted strain with an other one, then we know for sure that dapA deleted strain can be rescued by a strain producing DAP

Recombination frequency measurements

We discussed in the design process the question of the recombination frequency. We would like to have a tunable recombination frequency in order to find the optimal one, which should be somewhere between 0 and 50% of recombination per generation.

We should be able to tune the recombination frequency by modulating the Cre recombinase expression. In order to do this, we put the Cre under the control of the pBAD promoter. We then wanted to characterize our "Cre generator device", and to make the relation between the expression level and the recombination frequency.

To do this, we used a strain (FX85, provided by Francois-Xavier Barre) having the construction lox-KmR-lox inserted into its chromosome. We first though of transforming this strain with our pBad-Cre construct (on an Ampiciline resistance plasmid), then inducing the expression with different level of arabinose, and finally spreading on selective plates (Amp or Amp+Kan). The ratio between the number of clones on the two types of plates should give us the recombination frequency. When we tried to do this, we did not get any clones on the Amp+Kan plates, even without Cre induction. But this doesn't mean that the recombination frequency is 100%. Indeed, if the recombination frequency is around 50% per generation or higher, no colonies can grow on kan plates.

This means that our Cre generator is quite leaky and gives recombination rates too high even without induction. In order to know exactly what rate we had, we performed an other experiments. FX85 strain was transformed with our pBad-Cre construct and directly after transformation, launched into liquid culture with either Amp or Amp+Kan. 100µl of the culture were regularly plated on LB+Amp, giving the following growth curves:

dapAp characterisation

pBAD characterisation

DGAT cloning and triglycerid synthesis in E. Coli

Modeling results