Notebook
20 colonies expressing GFP
2 No growth
2B > 100 colonies expressing GFP
3 ~ 6 colonies - no GFP (did not glow under UV)
3B > 20 colonies expressing GFP
4A No growth
4B Lawn LA plate No ARA
5A No growth
5B No growth
6A No growth
6B No growth
7 No growth
UV sources
See Notes on UV Spectra - all the hardware is documented there
UV kill curve experiments at CCL (Rikke)
Need to normalize bacterial cultures - need to get spectrophotometer online to calculate cell count based on optical density (OD)
Use drop spot plate method for plate counts - saves a lot on plates
Use 96 well plates and multipipetters to do dilutions
Genes
Jay found the Anabaena genes available on plasmids at Addgene - $65 each!
http://www.addgene.org/Christopher_T_Walsh/
Let’s order what we already know we want to get from IDT asap. No need to order everything at once.
codon optimized Anabaena genes (with separate promoters / in separate biobricks?)
Nostoc final enzyme
GFPuv with strong promoter, strong RBS, transcriptional terminator
Need to decide which exact E.coli strain to use with RCA (especially whether or not it should be a RecA / DNA repair mutant!)
DH5 alpha sequence http://www.ncbi.nlm.nih.gov/bioproject/205928
Fundraising
Indiegogo editable https://www.indiegogo.com/projects/biosunblock-looking-at-sunscreen/x/6697587#/story
Need to finalize
FYI regarding images for the Indiegogo campaign: Instead of buying stock photos, look for images on Google Images that are labeled for reuse: http://screencast.com/t/bnrtTRvhBX
EELSI
Would be great to have 1-2 people who are dedicated to this. Join slack channel if interested.
Elizabeth (eapitts@ncsu.edu), James (jwantuck@gmail.com), Phil (phillipwantuck@gmail.com), Vikram (vikram.s3@gmail.com)
**email invitations to slack coming shortly
why is paba banned in EU
damage to coral reefs
nano/Australia
AddGene licenses not compatible w biobrick licenses?
Meeting 8/1/15
Attendees:
BioCurious: Maria C., Adarash, Phil, David Hou, Leo, Daniel (age 11), April, Zack (visiting from Atlanta EE), Jay Hanson, James, Johan, Meenakshi, Priyanka
CCL: Patrik, Andrew, Sean
Zoom: Rikke, Shreya, Antonio, Vardhaan, Victoria
Jamboree
Who signed up to go?
Patrik, Advait, Eri (may go as media)
Fundraising
- Did we decide on platform? Indiegogo
- Need a logo - April
- Budget: $6000 (minus pledges donated to team)
- Thank you, Stickers, Tee Shirt
- Research color changing beads in UV, tee shirt that changes colors in sunlight
- Quantum dot jewelry research as a stretch goal
- Make a gadget with UV LED as well
- Small GFP kit with plasmid $100
UV spectra
- mycosporine-glycine + shinorine/porphyra-334 + GFP covers UV spectrum nicely
- UV lamps for pet reptiles may be good choice for cheap broad spectrum UVA/B (add some leds for near UV?)
LEDs at 395-420nm are very easy to find at any electronics store (Radio Shack, Fry’s,...), but barely qualify as UV
- UV LEDs http://www.qphotonics.com/UVTOP-LEDs/
- 395nm LEDs http://www.ebay.com/itm/like/321364837871?lpid=82&chn=ps
- may need to hack one of our UV-specs to accept external light
- Start doing kill curve expts asap, on wild type e coli & mutator strain.
Start doing experiments at CCL on UV on Wednesday
Finalize IDT gene order
- Patrik can do codon optimization
- Which promoter & RBS to add? Or assemble everything from existing biobricks?
- Which flanking sequences do we need, for which assembly method?
- Do we include biobrick prefix/suffix?
Let’s use Genome Compiler - there’s a free iGEM version
Contact Minnesota team! Patrik will contact
Maria will contact iGEM about DNA distribution kit
Order free NEB kits
Decide what to order now
Assembly methods: https://j5.jbei.org/j5manual/pages/1.html
Gibson and InFusion: https://j5.jbei.org/j5manual/pages/22. Golden Gate: https://j5.jbei.org/j5manual/pages/23.htmlhtml
Biobrick Assembly: https://j5.jbei.org/j5manual/pages/21.html
GFP (BFP?) transformation today!
- GFP or BFP? GFP!
- just a warmup, or will we be able to use this construct together with the MAA pathway?
Take notes on all experiments in group notebook and person notebooks. Lookinto benchling.com
There are several deadlines this month. Be sure that your team meets the following:
August 07: Track selection
August 07: Title and abstract
August 28: Final Safety Form
Meeting 7/27/15
Attendees:
BioCurious: Eric H., Maria T. Chavez, Patrik, Eric A, Adarash, James, Rikke, Victoria, Dmitry, Jay, Erik I, Greg, Shreya, Michael, Samera, David, Meekakshi, Johan
Eric’s talk on RCA/RCR - Rolling circle amplification, Rolling circle replication
A way for viruses to replicate their DNA, a compact way to replicate their dna needing only 1 strand of DNA. Uses enzyme phi 29. A way to produce lots of DNA at a low temperature.
Need a circle, need an initiation point (a single stranded circle), 529 will initiate this at the lesion in a double stranded DNA. Phi 29 has a fidelity for doing this that is unreplicated so far from bascillius setellus (???)
RCA is not old technology, it was developed to make copies, for Sanger sequencing. To make a lot of copies of something that is a replication. Make a copy of a whole circle, strand replace and start again making a new circle of DNA. Pic of process - https://en.wikipedia.org/wiki/Rolling_circle_replication.
For our process is to NOT use phi 29 to introduce mutagenesis.
DNB is a DNA nanoball.
Eric today took M13 and put a nick in it to use it today. MB-BSM1 is an asymmetric cutting enzyme and will make a nick in the DNA either top strand (MB is the bottom strand).
Eric will post Protocol used for tonight.
Meeting 7/25/15
Attendees:
Biocurious: David M, Eric H, Jay, Phil, James, lafia, Meenakshi
CCL: Patrik, Andrew, Ramsel, Catherine (catherinesoneda@sbcglocal.net), Kye, Laurent (lorantto@gmail.com)
Zoom: Adarsh, David Hou, Shreya T, Rikke, Milo
Plasmid.com for preparing 1 mg of plasmid for $99 http://www.plasmid.com
When are we doing the GFP experiment?
Antonio Lamb (Amlamb@ucsc.edu), Jay, David, Rikke, David Hou, Shreya
Eric can teach a class on RCA: this Monday or Wednesday?
PIPE cloning papers: http://technology.sbkb.org/portal/page/334/
http://www.ncbi.nlm.nih.gov/pubmed/18988020
growing mutator strain without accumulating mutations in chromosome (see section 3.1): http://www.chemengr.ucsb.edu/~ceweb/faculty/daugherty/images/9-nguyen.pdf
Fundraising
Andrew, Catherine, Laurent, David Hou
Eric contacted NEB, will follow up with their iGEM coordinator and other contacts at NEB
adarsh: contact Agilent for directed evolution reagents. Agilent does not offer discounts for iGEM
Eric: just need cloning kit & buffers for RCA
Gene designs
Anyone to contact Minnesota 2012 team (Jay)
Link to Minnesota 2012 iGEM Team UV-Protective Compounds : https://2012.igem.org/Team:Minnesota/Project/UV_Absorption
Need to finalize gene designs, including any flanking sequences
Milo, Jay, Patrik
Look into possible DNA assembly methods:
Gibson
Golden Gate
Golden Braid
Infusion Cloning - Clonetech
OTHER GENES:
UV sensitive promoters? (see also SulAp)
reporter genes?
UV spectra and hardware
mycosporines absorb in UVB UVA (334nm)
GFP absorbs in UVA (395nm)
we have various UV illuminators (320nm, 365nm) - copy out some model numbers, and look up spectrum
Stratagene crosslinker
More research on killing E. coli with UVA, UVB, sunlight - what doses at what wavelengths
Rikke, Patrik
UV Sources:
At Biocurious: “Strategene crosslinker” - has replaceable / swappable UV bulbs, so will need to know what type of bulb is in there to determine radiation specs
Actual crosslinking is usually done with a very short-wave, very narrow-band UVC bulb (254nm), which would not be suitable for our purposes
Manual says it also has 302nm Midrange (UVB) bulbs (# 34-0042-01) and 365 nm Longwave (UVA) bulbs (# 34-0006-01),
UVP Cross linker Midrange 302nm UV:40 watts
http://uvp.com/crosslinker.html Midrange 302nm UV:40 watts
http://www.uvp.com/pdf/302wf.pdf
http://www.uvp.com/pdf/302.pdf
Meeting 7/18/15
Attendees:
Biocurious: Priyanka,jon,Eric Aker,Phillip, Audrey, April, Jay, David M, Adarsh, Johan
CCL: Patrik, Vishnu, Lorent, Andrew, Kye, Sean, Nathan
Zoom: Antonio, Rikke, Victoria, Advait
FUNDRAISING (experiment.com)
Maria, Patrik
INITIAL EXPERIMENT
Antonio Lamb (Amlamb@ucsc.edu), Jay, David, Rikke, David Hou
Eric can teach a class on RCA
ORDER GENS & REAGENTS
Please fill out the biography form - https://docs.google.com/forms/d/1D_fcWZVLaq6O3WoKQCFjSdUaI6MODaEFjqO_hauagxU/viewform?usp=send_form
Fundraising:
- iGEM registration fee $4000, bio bricks kit, and IDT discount for ordering DNA
- NEB has gotten back to us and is NOT offering community labs the discount for iGEM ** We should follow up and talk to them about this
- Options for fundraising - Crowdsource funding through Indiegogo, Experiment.com, gofundme, sponsorships, self funding
- Need to build a budget, each subgroup to let us know a very general cost, and what materials we need ordered (so we can look into getting supplies donated)
Which genes do we need to order?
https://2015.igem.org/Sponsors/IDT *Teams will receive value equivalent to twenty 1000 bp gBlocks Gene Fragments in their local currency. Promotion expires September 24, 2015.
Page with into to gBlocks video: http://www.idtdna.com/pages/landing/igem-2015
1. Shinorine pathway:
mycosporine-glycine pathway from Minnesota 2012 team is NOT available; need to reorder! Anabaena variabilis Ava_3855 to 3858
We could also order Nostoc punctiforme NpF5597 to 5600 as an alternative, and codon optimized versions of both
The Minnesota team could not get Ava_3855 to work, but we've identified 9 other enzymes that can do the final step in the pathway
2. pABA biosynthesis
BBa_K137055 from Caltech 2008 (promoter + RBS + PabA; IN 2015 IGEM KIT!)
BBa_K909014 from Zurich 2012 (P + RBS + PabB + RBS + PabA + term; IN IGEM 2015!)
express antisense RNA for pABA consuming enzyme?
3. GFP
What is the most favorite GFP biobrick?
4. UV sensitive promoters
BBa_J22106 RecA SOS promoter (in stock, not on distribution)
What else?
5. Any reporter genes (other than GFP!?
Need someone to look into UV light spectrums - could look into what UV lights we should be using (LED's that are mostly UVA, broad spectrum UV light) What spectrum, what hardware, what level of elimination to kill e-coli, what has been used by other labs - ** Rikke
Directed Evolution Update -
ELSI - April and Audrey will dig more into the issues around PABA
TODO:
- directed evolution look up $ estimates for experiments w mutator strain vs RCA
- come up with concrete list of genes to order from IDT
- transfer all notes to Wiki: http://bayareaigem.herokuapp.com/
New People Email(CCL):
Andrew.g.elkins@gmail.com
lorantto@gmail.com
eic@mycopathologia.net
nathan.eisenberg@att.net
Document the dates you worked on your project.
What should this page have?
- Chronological notes of what your team is doing.
- Brief descriptions of daily important events.
- Pictures of your progress.
- Mention who participated in what task.
Inspiration
You can see what others teams have done to organize their notes:
