Team:SF Bay Area DIYBio/Notebook

• TEAM
• PROJECT
  • Description
  • Experiments & Protocols
  • Results
  • Design
• PARTS
  • Team Parts
  • Basic Parts
  • Composite Parts
  • Part Collection
• NOTEBOOK
• ATTRIBUTIONS
• COLLAB
• PRACTICES
• SAFETY

Notebook

Meeting 9/12/15

Attendees:
BioCurious: Sairah, Maria C., Audrey, Priyanka, David Hou
CCL: Patrik, Jackie, Kye, Sam, Geoff, Sam

ZOOM: Adarsh, Victoria, Sean, Rikke, Meenakshi
PARTS SUBMISSION!
We need to mail out at least one BioBrick part by Friday, Sept 18!
Also need to enter that part into the database beforehand
WIKI EDITING!
All hands on deck for editing the wiki:
https://2015.igem.org/Team:SF_Bay_Area_DIYBio
The wiki gets “frozen” Friday, Sept 18, so we have to upload EVERYTHING there first. Lorent has uploaded our logo
FUNDRAISING!
All hands on deck for getting us finding. Indiegogo is up please donate and spread the word! https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587#/story
Experiments:
Mycosporine transformation into HB101 THIS AFTERNOON AT BIOCURIOUS, starting 1pm
UV spectrum of crude mycosporine methanol extract Here's two protocols for extraction of MAAs from cyanobacteria for spectroscopic analysis. As you can see the first one is very simple: extract in 100% methanol in the fridge overnight, centrifuge, and take the supernatant: https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats Cyanobacterial cells were harvested by centrifugation, and MAAs were extracted in 2 mL of 100% high-performance liquid chromatography (HPLC) grade methanol overnight at 4 °C. The methanol extracts were centrifuged at 10,000 rpm for 10 min, and the supernatant was subjected to spectroscopic analysis between 250- and 700-nm wavelengths, using a double-beam spectrophotometer (UV-Vis 2900, Hitachi, Japan). http://maxwellsci.com/print/crjbs/v3-165-171.pdf
Extraction of 10 mg of dried Aulosira fertilissima in 2 mL 20% Methanol(gradient grade) at 45ºC for 2h . Centrifugation if necessary (10 min 10000 U). 1.5 mL of the supernatant was lyophilised. 2 mL 100 % Methanol was redissolved in the residue further vortexing followed by centrifugation was done. 1.5 mL of the supernatant was evaporated at 45ºC. 1.5 mL H2O was redissolving in the residue, again vortexing and centrifugation was done. Spectroscopic analysis of the supernatant was taken from 200 to 750 nm.
Mycosporine like amino acid (MAAs) (Garcia pichelz et al., 1993)
Step 1. Took 10 mg fresh biomass and were suspended in 20 ml of methanol (20%) and homogenized.
Step2. Covered all the test tubes with aluminium foil and were kept at 45℃ in water bath for 2hrs.
Step3. Centrifuged and filtered the supernatant through whatman filter paper (No.1).
Step4. The absorbance of filtrate were measured spectrophotometrically at many wavelengths (310, 320, 330, 332, 334 and 260).
E. coli cells may be even easier to extract, because cyanobacteria tend to have a stronger cell wall Directed Evolution Round 1?
Patrik will start writing up a protocol
Everybody who has done research on Directed Evolution should help out digging up published protocols.
4. Domestication of gene clusters into IGEM plasmid restriction digest and ligation reaction (Tuesday or Wednesday)
We need Pstl; did we get that in the biobrick cloning kit from NEB?
Total Participation in iGEM:
102: on google group igem registered: 14 (other), 16(student), 7 (instructors) - total 37

Meeting 9/5/15 2 WEEKS LEFT!!!!

Attendees:
BioCurious: Maria, Jay, James, Phil, Prag (biomedical research at Stanford), Sairah, Lawrence (CS), April, Audrey, David H.
CCL: Kye, Megan, Patrik, Patrick, Tom, Maureen
ZOOM: Advait, Adarsh, Shreya, Rikke, David, Victoria


UV Hardware:
Drops in the 340nm and 390nm range according to spectra
Experiments:
Rikke starting another kill curve experiment at 1pm at CCL
Jay: transformation in mutagenic strain successful; nothing grew in HB101 (including pGLO positive control).
Need to start doing experiments collecting UV absorption spectrum:
- Measure UV absorption spectrum of TSB (tryptic soy broth). As you heard from Rikke, there seems to be an issue with fluorescence and/or UVabsorption in the medium we've been using for the kill curve experiments.
- Measure E. coli UV absorption spectrum, with and without the pGlo plasmid. (a) in whole cell suspension (b) in cell lysate, (c) in crude protein extract. If we hope to be able to detect a change in UV absorption spectrum in GFP, how should we best measure the spectrum?
- Once we have a mycosporine producing strain, we'll need some way to measure its spectrum as well. Eventually, we'd like to separate the MAAs by HPLC, but just like with the GFP, we could generate a spectrum for a crude extract as well. Here's a few protocols for extracting the MAA-containing fraction with methanol:
https://www.researchgate.net/publication/263709935_Biochemical_characterization_of_sunscreening_mycosporine-like_amino_acids_from_two_Nostoc_species_inhabiting_diverse_habitats
Cyanobacterial cells were harvested by centrifugation, and MAAs were extracted in 2 mL of 100% high-performance liquid chromatography (HPLC) grade methanol overnight at 4 °C. The methanol extracts were centrifuged at 10,000 rpm for 10 min, and the supernatant was subjected to spectroscopic analysis between 250- and 700-nm wavelengths, using a double-beam spectrophotometer (UV-Vis 2900, Hitachi, Japan).
IDT Order:
Need to finalize gene order for Anabaena gene cluster - see https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp=sharing
Jay and Patrik will coordinate on finalizing sequence; can also run by Eric Harness and Wayne
Survey:
https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/viewform?usp=send_form are you concerned about effect of sunscreens on your own health on the environment?
difference between UVA, UVB environmental issues are mainly on coral reefs, not coastal environment. and mostly due to other compounds than PABA, TiO2 would you wear sunscreen more if there were a more natural alternative?
Indiegogo Campaign:
Let’s put the fundraiser under CCL
Anyone who wants to work on video can do so.
Poster:
After wiki we will concentrate
Wiki:
Due on September 18th - everybody should be editing the wiki!
Presentation:
-Advait presented a talk at the S4 conference, we can use that powerpoint as a template

Meeting 8/29/15 3 Weeks left!!

Attendees:
BioCurious: Maria, Jay, Audrey, April, Priyanka, Sairah, David H.
CCL: Rikke, Kye, Sean
Zoom: David, Adarsh, Victoria, David M.
UV Hardware
Leave as is but UV exposure is not killing bacteria even at 1 hour of exposure, going to try moving closer to lights, right now we are at 8”
Problem with kill curve experiment: Some plates with high UV exposure have more growth than plate exposed to lower amounts of UV. In addition, GFP expression across plates are inconsistent.
potential hypotheses:
UV light is not close enough to kill majority of bacteria.
Bacteria is under stress due to UV irradiation and does not use pGLO plasmid as it doesn’t seem to provide benefit. Note that we are irradiating bacteria with a wide UV spectrum while GFP only absorbs a fraction of it. The TSB media partially absorbs UV light, sheltering bacteria.
Need to get anabana transformed into ecoli onto plate and to CCL
Jay will send out operan again form addgene

At BioC today will miniprep out 5 of the plasmids from addgene today, wont do transformation today (need to get competent cells ready)
And then bring to CCL for kill curve checks
Assume we will do transformations at BioC on Tuesday @ approx 5:30pm to start will take about 3 hours
Patrik start cultures on monday and wednesday and experiments on tuesday and thrusday need to schedule someone to do cell counting within 24 not 48 hours afterwards at CCL
EELSI
Survey on sunscreens - April, Audrey, David, Kye, Priyanka,
Sunscreen research - Wiki - needs more work!
Fundraising - stalled for some issues.
Poster - After wiki
Presentation - Advait first pass

Meeting 8/22/15 - 4 WEEKS LEFT!!!!

Attendees:
BioCurious: Maria I. C., Priyanka, Audrey, Adarsh, David Hou, Jonathan R.
CCL: Patrik, Kye, Sean
Zoom: James, Advait, Lorantto, Rikke, Wayne
Deadlines:
-Safety form (final version) due august 28
-https://2015.igem.org/Main_Page
-Maria, Eric,
-Team banners due September 1st
-Final team rosters due September 11th
-Parts due September 18th (http://parts.igem.org/DNA_Submission)
UV Hardware
Made a frame to hold UV lamps
Spectroradiometer is working
Reptile lamp covers UVB really well
Supposedly 380nm LED array is really ~410nm - mostly blue visible light!
One of the nail curing lamps has nice broad peaks around 365nm and 400nm
Reptile lamps + nail curing lamp has reasonable match to solar UV spectrum
Kill Curve Experiment
Coiled UV lamp in nail curing lamp may be burned out? Only designed to be on 1min at a time. Had to do without. Need to do better job at normalizing initial cell density. pGlo overnight culture had far fewer cells to start with. experiment notes: https://docs.google.com/document/d/1Fio3o6M9dsEPUZZ7Ig5h4nN_9p9mL4UdoYhWkxI7lF0/edit?usp=sharing
Need to pour another stack of quartered plates (8 left, pour 20 more with LB agar)
At CCL this week approx 8pm to 11pm?
Monday - figure out UV setup and start overnight batch of liquid culture (Patrik, Sean)
Tuesday - Kill curve part 2 (any changes from the first run?)
Wednesday - someone make more culture
Thursday - Another run of Kill Curve
At BioCurious - need to follow up with Jay (Maria emailing)
Need to get our HPLC running - check with Jacob, Eric, Josiah
https://docs.google.com/document/d/11vkbB8V8bB9dVdSySJ5CqLpqHBNjUqMy10cNifBWoeg/edit Genes
AddGene plasmids arrived; Jay plated them, and has copies for CCL
Still need to submit IDT order

description of Anabaena gene cluster:
https://docs.google.com/document/d/1AVc2TPD71psi-JXjWkUTxp79HHXIEM2vP6k7G7RxbWE/edit?usp=sharing
exclude restriction sites (used for standard biobricks assembly)
add any flanking sequences? Any restriction sites for Gibson? BioBrick prefix/suffix?
EELSI
Consolidated all documentation into one google docs: https://docs.google.com/document/d/1jt-huSIabQ6ZlggpiePb7-Lb4-eXLemOtNq-_lZqovM/edit?usp=sharing
Safety of working with UV light, safety precautions we have taken; include photographs of setup
https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen/x/6697587#/story
WIKI
Moving info over
template design for iGEM using logo etc.
Fundraising
change tagline on logo to Evolved Sunscreen on indiegogo
get BioC EIN
figure out which account to send funds to
Poster
layout and elements
Presentation
-Advait will also be presenting at S4 on Saturday (link?)

Meeting 8/15/15 - 5 WEEKS LEFT!

Attendees:
BioCurious: Maria, Adarash, Jay, Phil, James, Alberto (postdoc at Jbei), Lucan (masters at jbei), Daniel (phd starting at UC Berkeley), Deborah (biologist from Sao Paulo), Audrey (HS), Manakshi, priyanka
CCL: Sean, Kye
Zoom: David, Shreya, Victoria, Wayne, Manali, Advait
UV hardware
Sean: update on the spectroradiometer? Sean has purchased one off EBay and will see if he can direct pick up from seller in Vacaville. Will let us set up and monitor the kill curve experiments.
Victoria & Co: is the light box / stand ready to use?
email from Rikke with some issues we are having with the experiments
with UV lights will the heat be an issue - add a fan which will add in more contaminants
Rig modifications will be with Victoria at CCL today at 1pm
Kill Curve experiment
Do we have all the cultures, plates, reagents we need?
Have agar plates and bacterial cultures
bottle neck is - fan and specifically which wavelengths are hitting the bacteria
When do we start the experiment, and how long will it take?
will work on this afternoon and be ready to go this afternoon perhaps?
Email to get a protocol and set the date for the experiment (ask Rikke etc)
https://drive.google.com/open?id=0B2fL4PwIccWZfmRYc0lDdS1jWV9UeFdwME1EYmZxOG9EakFUZWNzS3pJczJCbU5QNElZSU0
Genes
8/21/15 Update from Jay (sorry can’t make today’s meeting)
The seven AddGene plasmids arrived. I streaked two sets of plates, one for CCL and one for BioCurious. FYI: Addgene used the Top10 E. coli cell line for delivering these plasmids. They are all Kanamycin resistant for selection. I will grow up some liquid cultures Saturday night and will be doing minipreps of the plasmids on Sunday afternoon starting around 3PM. Anyone interested in joining this miniprep festival is welcome!!
I’m in progress designing a GFP-UV gene with His-tag and flanking sequences in the Bio Brick format. As soon as the design is complete and checked we can order from IDT. Anyone interested in helping out on this is certainly welcome. Email me.
Link to Genome Compiler: https://igem.genomecompiler.com/igem_app
All: what else are we ordering from IDT, and when?
Has anyone looked at Jay’s gene cluster construct? Have someone else review
Need to order GFP with histags - Jay will design today in genome compiler folder
Fundraising
Indiegogo is ready we can launch today
Lets launch today!
Environmental/Ethical/Legal/Societal Issues (EELSI)
Elizabeth, James, Phil, Vikram: any updates?
needs cleanup but lots of research and info there
Community Engagement combined with fundraising (Reddit AMA on project) - to push fundraising - AMA in two weeks Saturday at noon led by James and Phil
Vikram (market survey how they are using the product) - Will have sample survey questions by next week
Wiki
Need to start populating the wiki with our final writeup on some of these topics (e.g. notes on UV spectra, etc.)
http://bayareaigem.herokuapp.com/
-
All experiments need to be documented every single day!
http://bayareaigem.herokuapp.com/index.php?title=Notebook

Everybody should submit a team bio: http://bayareaigem.herokuapp.com/index.php?title=Team
Maria will updates with submitted bios.

Meeting 8/8/15

Attendees:
BioCurious: Maria, Eric H., Daniel C, John McG, Zack, David H. (HS), Vikram, Manali (molecular Biologist), James (MD), Phil (materials scientist), Jonathan R. (yoga instructor)
CCL: Patrik, Tom, Sean, Elizabeth (visiting from NC research triangle iGEM team)
Zoom: Advait, Shreya, Vardaan, Rikke, Wayne, David M, VIctoria, Milo
Safety Training!
Everybody who wants to work in the wetlab needs to take the online safety quiz!
CCL:https://docs.google.com/forms/d/1n_R0MB1xJCMgdaEv1fklwtv-BRY0WmaPWhVQnNSg9cs/viewform
BioC:https://docs.google.com/forms/d/16m9xRfmq0obBe_aNBZwtNxUME_vDjVUieloK_jI6DVA/viewform
(You only need to take one - they are essentially identical. Please send us feedback if anything in the quiz is confusing)
Anyone working in CCL also needs to be a member
CCL will offer anyone on the iGEM team free or discounted membership.
Apply for sponsored membership here.
Or become a full paying member of CCL here.
Experiments update
See http://bayareaigem.herokuapp.com/index.php?title=Notebook
pGLO transformation at BioCurious (Jay)
The transformation we did on Saturday was successful. We got some transformants on plates 1B, 3B and 2B expressing GFP. I attached images showing all the plates and the GFP glowing under UV (let me know if you need a higher res image). Below is a table of the plate by plate outcomes. We used pGLO plasmids from different sources and some were over a year old so we expected that not all would successfully transform. Thanks to everyone who helped out on Saturday!!
I'll streak an HB101/pGLO plate for CCL. I plan to do a miniprep to purify the pGLO plasmid on Thursday night starting around 7pm if anyone wants to participate. Patrik will be at Biocurious on Thursday night and he can pick up a plate with the transformed HB101 and some plasmid to take back to CCL.
Plate Observation
1 Lawn LA plate no ARA
1B > 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, James, Phil, Vikram
**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, 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
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/

Document the dates you worked on your project.