Hello all!
I am working as an instructor to a HS iGEM team and as a part of their project I am helping them design bioluminescent yogurt. I have been through the previous thread and all the blog posts attached to the thread, but found that most of the ideas were lacking references and supportive literature. So, this is what i have proposed after going through the literature, your inputs and help will be much appreciated! :))
- Acetaldehyde: Acetaldehyde can react with luciferase complex to produce bioluminescence. Since acetaldehyde is produced by lactobacillus and streptococcus thermophilus in abundance, it will be an ideal substrate for the luciferase to chew-on. It is also among the most abundant chemicals on Earth and will help reducing the size of our construct from luxCDABEG to luxABG.
- Acidophilus: I am planning to work with acidophilus for the following reasons:
- Peroxide tolerance: Is more tolerant to H202 in compare to bulgaricus and several other species. Since bioluminescence will require aerobic conditions, the yogurt bacteria's will produce plenty of peroxide.
- Research: The bacteria has been thoroughly studied.
- Cost: My friend is making it available to me for free. He has been doing some research before.
- H202: Since acidophilus is H202 producing, the H202 can serve as an oxidizing agent required to form the flavin-peroxy intermediate. (Not sure!)
- NAD(P)H2.FMN oxidoreductase: It is found in some species of acidophilus. I can't find the literature for this, but I do happen to have read it somewhere. Also most of the L.casei species do have this enzyme so I believe we if required we could even add the particular enzyme to the construct.
Problems:
- Cathal in his blog post is favoring a linear transfer, but most of the literature is strongly against transformation with linear DNA. So, now I am confused in choosing between a plasmid transfer with chemical/ natural competence or a linear transfer?
- I am searching for a constitutive promoter for the purpose and would really appreciate any help with this.
- Is there anything which I am missing or might have not looked over. Your suggestions please! :)
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