From 2007.igem.org

Revision as of 09:21, 11 October 2007 (view source) Shollingshead (Talk | contribs) ← Older edit		Revision as of 09:32, 11 October 2007 (view source) Shollingshead (Talk | contribs) (→pTG262-Pbad-RFP construct) Newer edit →
Line 20:		Line 20:
	====pTG262-Pbad-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'

---

Revision as of 09:32, 11 October 2007

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.

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

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'