Team:Bielefeld-Germany/Results/Characterization/Exemplary results DM

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Characterization of mutated BBa_K389001 (exemplary)

As described in the section dealing with the theory of the screening system, colonies growing on the agar plates with 100 µg mL-1 kanamycin must either be induceable by one of the tested substances, or had received a constitutively active VirA variant. When we actually performed the subsequent experiments (EP PCR, cloning, transformation and primary selection) we were surprised to find many (approx. 100) positive clones within few days. To test which of substance in the inductor mix was able to active the VirA receptor, the clones were plated on selective agar plates with just one inductor at a time. To control if there might have occurred some mutations that lead to constitutive active VirA variants, all the bacteria were also plated on agar with kanamycine, without any inducer.

The results were hard to interpretate, as there was no clear cut-off but a broad spectrum of bacteria growing little, medium and fast on different plates. In the end we selected 23 promising clones that showed a slight tendency to be induced by capsaicin. The other substances (homovanillic acid, 3-O methyldopamine and dopamine) were excluded at this point of time, in order to focus on the molecule showing the best tendencies on the described selective plates.

For further analysis, we extracted the 23 promising VirA variants and changed the read out from kanamycin resistance to the expression of quantifiable luciferase procedure was described in the (theory and protocol section). Following we cultivated the bacteria in shake flasks and tested the response to induction with capsaicin, acetosyringone and without any inductor.

In the following measurement of luciferase we saw that the enzyme accumulated in all cultivations, including the cultures without any inductor. For quantitative analysis we calculated the production rate of luciferase in all cultivations. In order to compare the rates with the basal transcription in the native system, we divided all observed production rates by the luciferase production rate when using the native VirA without any inductor. The exemplary results of one clone with altered virA are shown in the following figure.

Figure 4: Luciferase production rates of the exemplary clone “21” are shown under conditions of induction with acetosyringone (21-A), capsaicin (21-C) and in uninduced state (21-U). The right bar indicates the production rate of the native system without acetosyringone (212-U), all the values in the figure had been normalized to.

On the left side of the figure the luciferase production rates of the exemplary clone “21” are shown under conditions of induction with acetosyringone (21-A), capsaicin (21-C) and in uninduced state (21-U). The right bar indicates the production rate of the native system without acetosyringone (212-U), all the values in the figure had been normalized to.

The interpretation of values shows that the receptor cascade in clone “21” results in an equal expression of luciferase whether there is any inductor (acetosyringone, capsaicin) or not. The production rate of the enzyme is in all those cases on a similar level. Moreover, these rates are two times higher than the value calculated for the native system without induction.

Taking all these observations into account it gets clear that the virA variant in clone “21” has lead to a constitutive expression of the reporter gene, that cannot be influenced by any tested inductor. These results were found for all further investigated clones. Consequently, none of the mutated variants of virA have been submitted to the registry.

Although were got the described negative results, the potential of our screening system should be pointed out. Within few days we mutated thousands of bacteria and easily selected many bacteria showing an altered, up-regulated receptor system. The techniques itself is thereby fast and appropriate when a screening system with a high throughput is necessary.