Edinburgh/Yoghurt/Proof of concept

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Revision as of 18:17, 26 October 2007

Introduction | Applications | Design | Modelling | Wet Lab | Proof of concept | References

In order to test the feasibility of gene expression in Lactobacillus and the possibility of making self flavouring yoghurt, we required a proof of concept.

As both the pigment and flavour pathways are rather complex and will require modification and optimisation before their expression in yoghurt we planned to use the much simpler Red Fluorescent Protein (RFP) gene as a proof of concept.

So far we have managed to transform a number of RFP-pTG262 vectors containing a variety of promoters into E. coli and also Bacillus subtilis, a gram positive bacterium.

Fig. 1: E. coli which has been successfully transformed with the pTG262-pLac-RFP construct

Fig. 2: E. coli which has been successfully transformed with pTG262-Ptet-RFP construct

We chose to initially transform Bacillus subtilis with our proof of concept vectors over Lactobacillus for two reasons:

• 1. the bacterium is naturally competent at certain stages of its life cycle and therefore has a much simpler transformation process.
• 2. members of the French lab have previous experience of transforming Bacillus subtilis.

pTG262-PLac-RFP construct

We have inserted the lactose induced RFP gene into the pTG262 vector

• this vector was then transformed into E. coli
• growth on IPTG-Xgal media resulted in RFP expression and production of red colonies. These can be viewed in figure 1

pTG262-Ptet-RFP construct

We ligated the Ptet induced RFP biobrick into the pTG262 vector. We then transformed the vector into E. coli.

RFP synthesis was observed.

Colonies can be viewed in figure 2.

pTG262 expression in Bacillus subtilis

After the successful expression of both the pTG262 RFP constructs in E. coli, we decided to determine if pTG262 could be stably maintained in Bacillus subtilis.

To do this we transformed both the pTG262 and Plac-RFP-pTG262 vectors into Bacillus subtilis.

Figure 3 depicts the successful transformation and expression of pTG262 in Bacillus subtilis.

This is determined by the ability of Bacillus to grow on chloramphenicol plates. No growth was observed on negative control plates.

Unfortunately RFP was not expressed from the promoter, as shown by the colonies being white rather than red. We are attributing this to the Bacillus transcription machinery (particularly RNA polymerase and its associated sigma factors) not recognising the promoter.

Fig. 3: Bacillus subtillis which has been successfully transformed with pTG262-plac-RFP vector

Fig. 4: E. coli which has been successfully transformed with lacz-pTG262 containing vector (blue colonies)

Introduction | Applications | Design | Modelling | Wet Lab | Proof of concept | References