Edinburgh/Yoghurt/Proof of concept

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Revision as of 17:25, 25 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 are going to use the much simpler 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 Bacillus subtillis, a gram positive bacterium.

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

1. the bacterium has a much simpler transformation process

2. members of the French lab have had successfull results from when they previously worked with 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

Further experimentation

• transform pTG262-Plac-RFP vector into Lactobacillus bulgaricus and Steptococcus thermophilus
• plate colonies onto IPTG-Xgal media and see if RFP is expressed
• introduce LAB with pTG262-Plac-RFP vector into yoghurt started culture & make yoghurt
• see if yoghurt produced turns red

Presently we are transforming the LAB with the pTG262-Plac-RFP vector

Transforming Bacillus subtillis with pTG262 vector

E. coli colonies successfully transformed with the pTG262-pLac-RFP construct

• we have managed to transform the pTG262 vector into Bacillus subtillis sucessfully
• colonies of Bacillus growing on chloramphenicol plates can be viewed in Fig 1.

E. coli colonies successfully transformed with pTG262-Ptet-RFP construct

pTG262-Pbad-RFP construct

For sugar induced gene expression, we have decided to insert the arabinose indued Pbad promoter in front of the RPF gene in the pTG262 vector.

• vector will be inserted into E. coli
• construct will be for arabinose induction of RFP production (plate on arabinose containing media)
• if successful P-bad-RFP containing pTG262 vector will be transformed into LAB
• LAB with Pbad-RFP vector will be used to make yogurt
• we will then test for red colour production upon the addition of arabinose

Possible problems with sugar induction of flavour and colours, could result from leaky promoter. Where the flavour and colour genes are expressed in the absence of the sugar inducer resulting in the yoghurt being flavoured or coloured when its supposed to be 'plain'

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