Edinburgh/Yoghurt/Proof of concept

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[[Image:Edinburgh Uni Logo.jpg|50 px]]
[[Image:Edinburgh Uni Logo.jpg|50 px]]
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[[Edinburgh/Yoghurt| Introduction]] | [[Edinburgh/Yoghurt/Applications|Applications]] | [[Edinburgh/Yoghurt/Design|Design]] | [[Edinburgh/Yoghurt/Modelling|Modelling]] | [[Edinburgh/Yoghurt/Wet Lab|Wet Lab]] | [[Edinburgh/Yoghurt/Proof of concept|Proof of concept]] | [[Edinburgh/Yoghurt/References|References]]
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[[Edinburgh/Yoghurt| Introduction]] | [[Edinburgh/Yoghurt/Applications|Applications]] | [[Edinburgh/Yoghurt/Design|Design]] | [[Edinburgh/Yoghurt/Modelling|Modelling]] | [[Edinburgh/Yoghurt/Wet Lab|Wet Lab]] | [[Edinburgh/Yoghurt/Proof of concept|Proof of concept]] | [[Edinburgh/Future| Future Directions]] | [[Edinburgh/Yoghurt/References|References]]
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__TOC__
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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.
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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.
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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.
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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 Red Fluorescent Protein (RFP) gene as a proof of concept.
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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.
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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.
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[[Image:PLac-RFP-pTG262 Bacillus.JPG|thumb|200 px|'''Fig. 1:''' ''E. coli'' which has been successfully transformed with the pTG262-pLac-RFP construct]]
[[Image:PLac-RFP-pTG262 Bacillus.JPG|thumb|200 px|'''Fig. 1:''' ''E. coli'' which has been successfully transformed with the pTG262-pLac-RFP construct]]
[[Image:PTet-RFP-pTG262 Bacillus.JPG|thumb|'''Fig. 2:''' ''E. coli'' which has been successfully transformed with pTG262-Ptet-RFP construct|200 px]]
[[Image:PTet-RFP-pTG262 Bacillus.JPG|thumb|'''Fig. 2:''' ''E. coli'' which has been successfully transformed with pTG262-Ptet-RFP construct|200 px]]
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We chose to initially transform ''Bacillus subtillis'' with our proof of concept vectors over ''Lactobacillus'' for two reasons:
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We chose to initially transform ''Bacillus subtilis'' with our proof of concept vectors over ''Lactobacillus'' for two reasons:
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1. the bacterium has a much simpler transformation process
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*1. the bacterium is naturally competent at certain stages of its life cycle and therefore has a much simpler transformation process.
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*2. members of the French lab have previous experience of transforming ''Bacillus subtilis''.
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2. members of the French lab have had successfull results from when they previously worked with ''Bacillus subtilis''
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We have inserted the lactose induced RFP gene into the pTG262 vector
We have inserted the lactose induced RFP gene into the pTG262 vector
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* this vector was then transformed into E. coli
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* this vector was then transformed into ''E. coli''
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* growth on IPTG-Xgal media resulted in RFP expression and production of red colonies, these can be viewed in figure 1
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* red colonies were produced on standard growth media. These can be viewed in Figure 1.
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We ligated the Ptet induced RFP biobrick into the pTG262 vector.  We then transformed the vector into ''E. coli''.
We ligated the Ptet induced RFP biobrick into the pTG262 vector.  We then transformed the vector into ''E. coli''.
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RFP synthesis was observed when ''E. coli'' colonies containing the vector were incubated on tetracycline containing plates.
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RFP synthesis was observed.
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ptet incuded RFP ''E. coli'' colonies can be viewed in figure 2.
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Colonies can be viewed in Figure 2.
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==pTG262 expression in ''Bacillus subtilis''==
==pTG262 expression in ''Bacillus subtilis''==
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After the successful expression of both the pTG262 RFP constructs in ''E. coli'', we decided to determine if pTG262 could be stably experessed in ''Bacillus subtilis''.
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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''.
To do this we transformed both the pTG262 and Plac-RFP-pTG262 vectors into ''Bacillus subtilis''.
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Figure 3 depicts the successful transformation and expression of pTG262 in ''Bacillus subtilis''.
Figure 3 depicts the successful transformation and expression of pTG262 in ''Bacillus subtilis''.
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This is determined by the ability of ''Bacillus'' to grow on chloramphenicol plates.
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This is determined by the ability of ''Bacillus'' to grow on chloramphenicol plates. No growth was observed on negative control plates.
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Unfortunately RFP was not expressed from the pLac promoter, as shown by the colonies being white not red.  We are attributing this to the ''Bacillus'' transcription machinary (particulally RNA polymersase and its associated sigma factors) not recognising the pLac promoter.
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We also transformed the a lacz-pTG262 vector into ''Bacillus subtilis''.
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When the transformants were grown on an IPTG containing media colonies turned blue, indicating that lacz (beta-galactosidase gene) was successfully expressed in ''Bacillus subtilis''.
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The results of this experiment may be viewed in figure 4.
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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.
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[[Image:PTG262 Bacillus colonies.JPG|thumb|left|'''Fig. 3:''' ''Bacillus subtillis'' which has been successfully transformed with pTG262-plac-RFP vector|300 px]]  [[Image:PTG262-lacz Bacillus.JPG|thumb|300 px|'''Fig. 4:''' ''Bacillus subtillis'' which has been successfully transformed with lacz-pTG262 containing vector (blue colonies)]]
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[[Image:PTG262 Bacillus colonies.JPG|thumb|left|'''Fig. 3:''' ''Bacillus subtillis'' which has been successfully transformed with pTG262-plac-RFP vector|300 px]]  [[Image:PTG262-lacz Bacillus.JPG|thumb|300 px|'''Fig. 4:''' ''E. coli'' which has been successfully transformed with lacz-pTG262 containing vector (blue colonies)]]
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[[Edinburgh/Yoghurt| Introduction]] | [[Edinburgh/Yoghurt/Applications|Applications]] | [[Edinburgh/Yoghurt/Design|Design]] | [[Edinburgh/Yoghurt/Modelling|Modelling]] | [[Edinburgh/Yoghurt/Wet Lab|Wet Lab]] | [[Edinburgh/Yoghurt/Proof of concept|Proof of concept]] | [[Edinburgh/Yoghurt/References|References]]
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[[Edinburgh/Yoghurt| Introduction]] | [[Edinburgh/Yoghurt/Applications|Applications]] | [[Edinburgh/Yoghurt/Design|Design]] | [[Edinburgh/Yoghurt/Modelling|Modelling]] | [[Edinburgh/Yoghurt/Wet Lab|Wet Lab]] | [[Edinburgh/Yoghurt/Proof of concept|Proof of concept]] | [[Edinburgh/Future| Future Directions]] | [[Edinburgh/Yoghurt/References|References]]
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Latest revision as of 20:26, 26 October 2007

Edinburgh Uni Logo.jpg Introduction | Applications | Design | Modelling | Wet Lab | Proof of concept | Future Directions | References

Contents


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
  • red colonies were produced on standard growth media. 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 | Future Directions | References

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