OBJECTIVE: Hack a cell culture to produce bioluminescent light using luciferase
participants: anitha, cameron, jason, jessabella, kalpith, kim, matheo, michael, patrik d, patrick s, scott, steve
As a start, the pGLO infrastucture from the Green Fluorescence Protein (GFP) Experiments could be used, substituting the GFP gene with the luciferase gene, using the presence of arabinose to start gene expression and therefore luminescent activity. The 2010 E. glowli iGEM project from Cambridge produced a construct that would be an excellent starting point: http://partsregistry.org/Part:BBa_K325909
Further down the road we can think of other ways to activate the bioluminescent behaviour, add our own "bioLOGIC"
Our weekly meeting at BioCurious is on Mondays, 7:30-10pm
Also make sure to sign up for the mailing list if you want to stay informed:
https://groups.google.com/group/biocurious-bioluminescence
For basic information on bioluminescence, click here.
CURRENT PROJECTS:
Lux Operon Engineering
We're exploring two different approaches right now:
-
-
- cannot be engineered; focus on optimizing growth conditions etc.
Things to do:
- E. glowli LuxBrick plasmid
- Get plasmid and biobricks
- Transform into E. coli
- Compare with our pLux E. coli
- Build E.glometer - DONE
- Build Temperature sensor (optional)
Medium Term Goals:
Glows longer
Glows brighter
Novel colors
Novel switches/promoters
Long Term Goals:
Random mutagenesis
Yeast , cyanobacteria, Other Organisms
L-Cysteine firefly luciferin recycling pathway
Needs:
Temp Meter
Fluorescence Meter
Next Meeting will be:
Next Monday at 7.30pm @ Biocurious
(new people are more than welcome to join in any time)
Resources:
The 2010 E. glowli iGEM project from Cambridge - check out some of their great videos:
Project overview: https://www.youtube.com/watch?v=nvE2IMTiWMY
The Bacterial Bubble Lamp: https://www.youtube.com/watch?v=tUFscEVK5Ks
The "Terrific Science" website has some great lesson plans on DNA for High School/College level. Including:
AddGene has some plasmids with the Photorhabdus luminescens genes:
Another Vibrio lux plasmid is pVIB, formerly called pJE202 here: EngebrechtCell83.pdf
Carolina Biological Supply has the pVIb plasmid, and kits:
Isolation of bioluminescent bacteria (may require BSL-2 facilities, officially):
Bioluminescence Web sites
Bonnie Basslers TED Talk on "The secret social lives of bacteria"
http://www.youtube.com/watch?v=TVfmUfr8VPA
(patrick: this talk inspired and motivated me to start this project. Taking it as a fact that bacteria and cells in general do communicate with the cellular network ouround them and are "aware" of their surroundings, the following questions and ideas arise: How can we "communicate" with cells? What will cultures do when you give them "lifestyle" choices?
So I am interested in creating heterogenous environments for cell cultures, instead of giving them just their desired medium and letting it sit in their desired temperature.
Ideas:
Petri dish with gradient temperature difference (controlled with open source and hardware e.g. with arduino)
Also create experiment environments where cultures can choose/ "grow towards" different:
- Nutrients
- Toxins
- Different wavelength of light
- environments with competing culture strains
- absence of liquid / nutrients,)
So to recap: find out their "Likes" and "Dislikes", survival strategies, communication....
Siouxsie Wiles Animation on Vibrio Fischeri
http://youtu.be/KCobcWsYOS8
Common luciferase systems in organisms
http://www.lifesci.ucsb.edu/~biolum/chem/detail1.html
Paper on "bacterial Bioluminescence"
http://www.photobiology.info/Lin.html
If we want to extract the lux operon from Vibrio directly, and make our own plasmid from scratch, ATCC Strain Number 7744 might provide an inexpensive ($40) source of the lux genes. Need to check GenBank.
http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=7744&Template=bacteria
Cyanobacterial iGEM projects:
Examples
- Philips Design Microbial Home (similarly something IDEO is thinking about)
-
Bio-light : "The bio-light uses different biological technologies to create ambient light effects. The concept explores the use of bioluminescent bacteria, which are fed with methane and composted material (drawn from the methane digester in the Microbial Home system). Alternatively the cellular light array can be filled with fluorescent proteins that emit different frequencies of light."
Perform PCR Reactions
Comments (9)
Lou Hom said
at 10:58 pm on Jan 9, 2012
When you say luciferase, do you mean the luxCDABE operon from Vibrio?
Patrick said
at 11:29 pm on Jan 9, 2012
Actually I am quite new to the field and am not sure how exactly it will work, I only got this vision and commitment and trying to make it real now. The very first Meeting will be next Monday, would be great if could come and share your thoughts. People who want to help lead the project are more then welcome to do so, someone with a background in this field would be highly appreciated.
Patrik D'haeseleer said
at 11:28 pm on Jan 9, 2012
Vibrio would be the easiste place to start, presumably. There are a number of different luciferin/luciferase systems in nature, but I'm not sure which all are commercially available. We could have a little internal competition on which team can produce the strongest bioluminescent E. coli.
For a "nightlamp", I do think we eventually want to move to some system that is a bit more independent of human intervention. If we could engineer bioluminescence into a cyanobacteria, we could potentially have a closed jar that photosynthesizes during the day and bioluminesces at night. Maybe add it into a little biosphere to get a completely closed system...
Patrick said
at 5:31 pm on Jan 10, 2012
Talking about "Vibrio", do you mean taking a Vibrio Fischeri culture from out there and just experimenting with it, or take the specific gene from that strain and put it into E.Coli?
I will add a source which is a manual to get Vibrio Fischeri from a Herring Fish. I only find german sources, so I hope you dont need a Herring from the east sea. I will check in japantown and chinatown if i can find a herring for this experiment.also I heard there are a lot of cultures in the ocean, too, just here at the beaches. If anybody knows how to get bioluminescent cultures let us know!
The cyanobacteria which harvests sun energy during the day and emits light through bioluminescence at night is one of the most awesome Ideas I've heard, that would be more than amazing if you could realize that.
Patrik D'haeseleer said
at 6:22 pm on Jan 10, 2012
We should definitely *not* deal with environmental sources for now. There are plenty of validated and characterized luciferase systems available, some Vibrio species are toxic or pathogenic, and we can't isolate environmental bacteria without BSL-2 capabilities - which we don't have yet.
A number of iGEM projects have worked with luciferase systems before, and we should probably start where they left off (or start by redoing what they did, as an exercise). The 2010 E. glowli project from Cambridge seems like a good resource:
http://2010.igem.org/Team:Cambridge
Here's a good overview of the different luciferin/luciferase systems out there:
http://www.lifesci.ucsb.edu/~biolum/chem/detail1.html
Patrik D'haeseleer said
at 6:37 pm on Jan 10, 2012
Looks like this is the BioBrick to start with, containing the LuxA,B genes for the luciferase, and the LuxC,D,E genes for luciferin synthesis, controlled by an arabinose promoter (pBad):
http://partsregistry.org/Part:BBa_K325909
"As of October 2010 we believe this is the first and only BioBrick to emit light in normal E. coli strains without the addition of any external substrate. "
Patrick said
at 11:31 pm on Jan 9, 2012
I just realized that this thread is just a page and not a folder like the Bioprinter Project. i am using this software for the first time today, does anybody know how to change this?
Patrick said
at 1:17 am on Jan 17, 2012
ok thank you, someone solved it :-)
Patrick said
at 1:29 am on Jan 17, 2012
Thank you everyone for the participation this evening at our kickoff meeting for the bioluminescence project! Thanks for sharing your ideas and inspiration! This project will be awesome... specially when we turn the lights off... :-)
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