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

Notebook

The following are the notes from our weekly organizational meetings:

Meeting 11/14/15

BioCurious: Maria, Jay, Sairah, David H.

Zoom: Advait

CCL: Patrik, Rikke,

Indiegogo - emailed about tee shirt sizes, will order shirts next week to ship or hand out.

Patrik - get Jay a check

Rikke - going to check incubator at CCL, see what needs to be done to get us up and going again Ava 358 to 356 in liquid culture which might be viable if it were streaked

Rikke will come in during the week (wed?) to try restreak from liquid cultures in incubator

Directed evolution piece - what do we need to get to Eric to get it done?  need to do a plasmid prep

---

Meeting 10/31/15

BioCurious: Maria, Sairah

Zoom: Advait, Audrey, Rikke

CCL: Patrik, DJ

Indiegogo

iGEM wiki

edits can be made until Nov 20

Wiki: https://2015.igem.org/Team:SF_Bay_Area_DIYBio

iGEM presentation: https://drive.google.com/open?id=1FvV12vXe0GxkEbP3Ved9sdExBqf4ekycNbJg9QtXxsc

iGEM poster:

https://drive.google.com/open?id=0Bx0m7X6dHC0mOHdSNENTZURzdEk

Experiments

BioC

Jay, Sairah and Meenakshi worked on lab work this week on Thursday and Friday night

They cultured the Ava genes and the positive control pglo and negative, of “no plasmid.” Did 2 batches of ligation product: one at 16C-30 minutes/80C-20 minutes on 10/29/15 thurs, transformed right after. Batch #2: 4C/Overnight, transformed after 21 hours on 10/30/2015 friday.

• Neither batch has colonies

• find or order more NEB bio brick kit

CCL

State of the lab at CCL is holding back doing more experiments.  Rikke will be doing more organization.  Advertise that we will be doing a cleanup on Tuesday if folks can help out. Part of the lab should be useable by Wednesday

Need to make glycerine stock. Do a simple class on how to make basic 101 lab techniques like making glycerine stock - Wayne can teach (but needs a week notice)

-https://www.addgene.org/plasmid-protocols/create-glycerol-stock/

• need to revive the stock
• need to do the RCA (talk to eric about it)
• get plates and do a mini prep (if something grows can put on glycerine)

Email Wayne for a date to teach a short session at CCL on how to make glycerine stocks

(other classes needed, making media, pouring plates, making liquid cultures, basic bacterial culturing)  Rikee: liquid media, plate pouring, streaking

https://www.washingtonpost.com/news/energy-environment/wp/2015/10/20/after-sunscreen-protects-humans-it-massacres-coral-reefs/

---

Meeting 10/17/15

BioCurious: Maria, David H.

Zoom: Meenakshi

CCL: Patrik, Rachel, Maureen

Indiegogo

Put account information and money should have arrived. Patrik will check bank account

$1564 total raise with 19 backers

% going to indiegogo

Need a list of everyone that needs paying back and expenses thus far

Reimbursement will come through CCL via Patrik

Need to reach out to funders to get tee shirt sizes etc.

Need to send out final email thanking folks and what next steps are etc.

Stickers: 2x3”?

Experiments

BioCurious

Tried transformation, nothing came out, no colonies on plate.

Making new competent cells re-trying on Thursday evening

going to start with re-ligating then trying the transformation again

Upcoming Experiments

Continue trying to retransform IDT parts at BioCurious

Try doing a Directed Evolution round at CCL? Need to check with Rikke.

Wiki

Need to update iGEM wiki with the figures from poster and presentation

OnGoing

Will reevaluate how many people are coming and how long to keep the project going on a bi-weekly basis

---

Meeting 10/3/15

BioCurious: Maria, Jay H., David H., Sairah

Zoom: Advait, Adarsh, Audrey, Victoria, Kye, Milo,Meenakshi

CCL: Patrik

iGEM recap

• We did well, poster looked good, got a bronze medal, missed things on the wiki (they formally check the boxes), gene design wasn’t on there, no page with colabs, weakest link was wiki)

• Judges liked it, didnt have a lot of questions,
• Easier for HS teams to put together funding and collab with a DIY lab
• Patrik wants to do an article for BioCoder about DIY labs experience with iGEM
• two meetings on Sat, discussion session about new tracks at iGEM (hardware, art, etc); large discussion on costs that not only will costs NOT go down might go down and might go to weed out weaker teams, don’t have a grant writer on staff, no discount to community labs), no formal process for grants for 3rd world countries etc.
• Alternative, join the Labs Program: program for labs to make biobricks and get parts $500/year - https://igem.org/Labs_Program
• DIY Bio labs should set up something for themselves aimed for DIY BioLab open to anyone HS or smaller colleges, lower costs.  If a hackathon make it a week long to get something accomplished. Many different possibilities.
• South American groups are forming their own iGEM alternative - Maria is contact point
• For community involvement: ask Citizen Schools, which offers a lot of established tech/STEM after school programs for lower-income/minority students, to maybe start an after school DIYBio/Introduction to Synthetic Biology- that way, involving students who may never otherwise experience this exposures
• Marc Juul is working with new BioBricks Foundation push, a node system for automated freezer to let labs network who has parts, opportunity for a challenge in lab automation
• Putting together mailing list for people interested in developing educational outreach programs with local schools. Willam&Mary team at iGEM had beautiful 80-page curriculum of simple classes. See also http://www.citizenschools.org/
• WIll hand out certificate for igem participation at CCL and BIoC

Reschedule meetings? Maria will send out a google poll  

Link for poll: http://goo.gl/forms/dr3Gacd862

Option 1: Stay on Saturday mornings

Maybe drop down to meeting every other week?

Option 2: Move to Mondays, every other week

Alternating with with Real Vegan Cheese

Option 3: Merge with Real Vegan Cheese meetings?

We have a good amount of overlap with the RVC team anyway.

Option 4: any other time slots?

DNA synthesis

• We may still be able to convince IDT to give us the remaining $1400 in DNA synthesis, but someone will need to negotiate with them - any volunteers?
• We have the Addgene plasmids, and the initial set of IDT genes - all native cyanobacterial sequence. That good enough? If anything, it should provide lots of room for improvement by Directed Evolution...

Website

We haven't done a very good job at showing to the general public what this project is about. The iGEM wiki we threw together at the very last minute is missing some major pieces of information. The iGEM wikis do attract a lot of traffic, so now that the wikis are unfrozen we should either clean it up or link out to whatever else we'd like to be the "front page" for this project (like we did for Vegan Cheese last year). We made some great diagrams for the iGEM poster and presentation that we can easily reuse for a kickass website…

Team - Advait, Sairah, Maria, Patrik, Rikke, Milo

Indiegogo

https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen

Extend deadline? Add materials from poster, presentation.

6 days for more funding

What should be our end goal for this project?

1. A statistically significant improvement in UV absorption? In E.coli UV resistance?
2. A publishable result?

Experiments

What is next? Are we ready to do a round of Directed Evolution?

Who/When/Where?

Next experimental work: At BioCurious; transformation of IDT genes

Regular work night at CCL on Wednesdays - Mention on Lab Night meetup description

Potentially doing lab work at BioC on Thursday nights led by Jay

Need to do RCA nicking, check in with Eric H.

---

Meeting 9/26/15 AT IGEM JAMBOREE

BioCurious: Maria, Pryianka, Sairah

Zoom: Victoria, Shreya, Kye

CCL: Tom

iGEM: Patrik, Advait, David, Milo

Questions from the Jamboree team:

• get protocol that was used for UV absorption spectrum: cells grown 20ml overnight in TSB, centrifuged, washed in saline 2x times, how much cell pellet vs 2ml methanol, pellet 10000rpm, collect supernatant
• how does UV irradiation level compare with sunlight? Need NUMBERS

• 1.5-2 mW/cm2 vs >15 mW/cm2 UVA+B light meter

• UV spectrum of other light sources (e.g. LED array)?
• Can we claim Maker Faire? Did we have anything on our project there? - We can claim we recruited members and educated about iGEM
• was RCA workshop open / on meetup? - it was for iGEM team members only not on Meetup
• Any more headshots? Yes - Maria will email them, some folks switched out what they have, I will send updated slide or I can send the emails with the pics
• Can we get a picture of people at BioCurious connecting over zoom, - we can try

iGEM labs ~$500 a year and get DNA distribution and can submit parts year round

TOm - at ccl  will send him info on project -

Indiegogo - add new reward of color changing beads, get more people to donate!

We will need experiment schedule.

Next week regular meeting time - then we will decide about going bi-weekly for meetings.

---

Meeting 9/19/15 ONE WEEK LEFT TO THE IGEM JAMBOREE

GOOGLE SLIDES: https://docs.google.com/presentation/d/1t0tGrPprF2e3KcYOzP06BX6zoozQeELeKH73cEZau24/edit#slide=id.gbbca6c085_1_0

Attendees:

BioCurious: Maria, Priyanka, Adarsh, David H., Sairah, Shree, Shreya

CCL: Patrik, Kye

ZOOM: Advait, Victoria, Rikke, Meenakshi

Parts and wiki submitted on time! Yay!

Our team presents NEXT SUNDAY in Boston

The presentation is probably the most important factor in judging, so we can still get any last-minute results in, over the next week!

Judging Forms (REOPENED!)

-Required for any special awards (such as outreach, entrepreneurship, etc)

-https://igem.org/2015_Judging_Form?id=1839

Experiments for the week (fluid depending on step before)

first round of directed evolution, taking Addgene plasmid (do we have any at BioCurious prepped by Jay? if not then Rikke will be doing a mini prep on liquid cultures) hand off to Eric who will do first step of error prone RCA (to give us mutated plasmids), another round of transformations with the plasmid, take those expose to UV light and then another round of plasmid prep, what ever survives will give us another round and run a gel, and then another round of RCA.

IDT Gene Design

ask Eric, Wayne (who else?) about issue regarding repeated promoters

Poster

Milo started on poster layout will go with ppt

ATTENDING IGEM IN BOSTON AND PRESENTING

(Please list when you will be arriving, and where you will be staying)

Patrik: Likely arriving morning of 25th; haven’t booked yet

Advait - Morning of 24th, Staying in Sheraton

David H.

Milo (Arriving Thursday morning, getting into Logan at 10am. Staying in Cambridge)

Presentation

David will put Advait’s into Google all members of the team will work on it

https://docs.google.com/presentation/d/1t0tGrPprF2e3KcYOzP06BX6zoozQeELeKH73cEZau24/edit#slide=id.gbbca6c085_1_0

Fundraising

https://www.indiegogo.com/projects/biosunblock-evolved-sunscreen

Minimum everyone donates $1, we need more folks contributing, spread the word

Survey

 To edit Survey:

https://docs.google.com/forms/d/19URbYIXXwWi9Hv4B7BM6-iEntHuiCIOcK3TGyA39sjI/edit

SHARE THIS LINK FOR SURVEY:

http://goo.gl/forms/pyqLgoP97Y

Lessons Learned

https://docs.google.com/document/d/1TvJ6YEQvp0s7oNeDIaJOSC0VLT_aC3IFNAEo4yH8sqc/edit?usp=sharing

We will meet next week for a shorter experiment update at 10:30am

---

Meeting 9/12/15 1 WEEK LEFT!!!!

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:

1. Mycosporine transformation into HB101

THIS AFTERNOON AT BIOCURIOUS, starting 1pm

2. 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

3. 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:

1. UV light is not close enough to kill majority of bacteria.
2. 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.
3. 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

1. 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

 

2. UV sources

See Notes on UV Spectra - all the hardware is documented there

3. 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, Kye, Laurent

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

1. http://www.uvp.com/pdf/302wf.pdf
2. 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, 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/