The Boston Open Source Science Lab

Notes from Jan 29, 2012 Meeting

Mon, 30 Jan 2012 10:41:30 -0500

BOSSLab Meeting
Jan 29, 2012 6 to ~9:30 PM Eastern

Attending:
Avery
Brian
Dan
Ian
Nancy
Rachel
Yoav

Updates:
Dinoflagellates:
It is difficult to get a good still image of the luminescing dinos. Exposure time is best ~5s.
Rachel is working on quantifying the luminosity and growth rate of the dinos.
Avery would like to investigate manipulating the dinos’ circadian cycle

Electrodeposition:
Ian is still looking for a lab to do the analysis. Next project would be to devise an enclosed deposit that fills with gas from the anode.

Glassware:
Rachel has started organizing glassware into Unambiguously Clean Glassware and Clean? Glassware. Labels are in the back of the yellow cabinet.

New stuff:

Regular meetings will now be held on the first and third Sunday of the month at 6 PM at SPROUT.

Bacteria Printer:
Nancy and Yoav want to make a ‘bacteria printer’ that deposits bioluminescent, fluorescent or brightly pigmented bacteria on an agar plate. She is starting to become familiar with stepper motors.

Experimental evolution of multicellularity:
Ian would like to attempt to replicate the recent emergent multicellular yeast artificial selection / directed evolution experiment listed here http://www.pnas.org/content/early/2012/01/10/1115323109
Expensive materials are S. cervisiae strain Y-55 and β-glucuronidase/arylsulfatase - let Ian know if you find an inexpensive or donation source

Glovebox / Laminar flow hood:
Dan would like to have an enclosed space to reduce sample contamination. Rachel knows someone who made a laminar flow hood using plastic, cardboard and HEPA filters. Avery noted that having an open flame (ex: alcohol burner) greatly reduces contamination.

Webcam:
Avery is working on getting us a streaming webcam. He’ll need the SPROUT router password; Rachel will reach out to Alec and Nagle

Miscellaneous:

SPROUT kitchen has been dismantled.
Propane tank is now in the shed in front of SPROUT.
We briefly discussed remote controlled rats, nudibranchs (voracious appetites), Elysia chlorotica and http://wtf-nature.livejournal.com/
Backyard Brains http://www.backyardbrains.com/Home.aspx has a kit that allows you to electronically control cockroach legs.
On our bench are are two blue cups containing fluorescent green fluid and UV LEDs in them. Please let us know if they are yours.
Avery is growing anemones. Avery would like to grow up some E. coli with GFP plasmid and extract the plasmids so we don’t have to buy them.
Brian has been working on weekend projects, but wants to team up on something longer
Dan donated three small reagent bottles and a bottle brush. Dan would like to replicate various fish DNA barcoding studies with local sushi.
Nancy is working with wireless sensors - Avery has some experience with the platform she is using.
Rachel wants to do a bioluminescent installation piece
Yoav is part of the MIT Media Lab
General advice: “Don’t eat it”

General Meetup Jan 29 @ 6 PM

Fri, 27 Jan 2012 07:37:39 -0500

Meeting Sunday January 29, 2012 at 6:00 PM

At Sprout (339R Summer St. Somerville, MA 02144)

Dinoflagellates Jan 20, 2012

Fri, 27 Jan 2012 07:34:00 -0500

Avery & Rachel’s dinoflagellates are doing very well. See the other photos here: http://stringeth.me/me/?p=82

Electrodeposition Update 2012 Jan 03

Mon, 02 Jan 2012 18:38:00 -0500

Lots of deposits

I hadn’t seen the setup in a week or two. I noticed power was 1.06 A @ 4.7 V - much higher than the 0.04 A target. I don’t know if this is due to power supply drift or someone changing the setting.

The solution was murky and there were lots of deposits:

View from above:

It seems the carbon from the anode went into solution. Here one can see the corrosion on the electrode:

A photo of the cathode deposits. They remind me of cave formations:

Some detail pictures:

The plan is to let the cathode dry out and send samples for analysis. I suspect the deposits are calcite, aragonite and dolomite - meaning calcium carbonate and magnesium carbonate.

Dec 4, 2011 Avery and Rachel autoclave glassware and synthetic...

Mon, 05 Dec 2011 11:39:00 -0500

Dec 4, 2011 Avery and Rachel autoclave glassware and synthetic saltwater in prep for growing dinoflaggellates

Notes from Oct 2, 2011 Meeting

Mon, 03 Oct 2011 08:46:00 -0400

BOSSLab Meeting
Oct 2, 2011 2 to ~5 PM Eastern

Attending:
Dan
Ian
Lasse
Meghan

Updates:
IPA bottled. Lots of prep work, sterilizing with iodine. Kind of messy. Initially stuck wrong end of siphon in fermentation bucket. Should be ready to drink Oct 23. Monetary contributions should be put in the Sprout donation jar.

Waste from recent projects bleached, pressure sterilized and put in landfill trash. Pressure sterilizer pressure still hard to control with propane burner. Autoclave bags got wet and ruptured - pressure sterilizer bleached and cleaned with Alconex. Rinsed emptied to public sewer.

Electrodeposition equipment brought in by Ian, along with 2-day deposits on 1/4” hardware cloth. Experiments using metal anodes - even surgical steel - resulted in the dissolved anodes. This is common in electroplating, and not surprising (bit did make for some colorful foam). Switched to carbon anode, which does not dissolve. Safety for next run (on power supply, not battery) will include voltage and amperage limits, an enclosed chamber (aquarium) and warning signs. Dan will research user manuals for Sprout’s power supplies and get a 10 to 20 gallon aquarium. Evaporation / loss due to electrolysis may be significant.

Lasse will be leading the webcam project. He is very interested in working with others on it. Plan is to use it to remotely monitor projects. Would be nice to use this to make a time-lapse video of the electrodeposition results. Avery has done this in the past.

Meghan will be leading our next transformation project. She’s interested in other’s ideas on the particulars for what traits to insert. Banana scent gene is available. Some folks at MIT are working on variable fluorescence expression based on electric current. Gabriel found an easy UV fluorescence transformation protocol: http://biotech.biology.arizona.edu/labs/transformation(TG).html

Meghan brought up plastic-digesting organism isolation as an alternative. Avery has some background in this. Charlie can supply protocols. Paul wants to help with this.

Lasse would like to lead an automated incubator, starting with setting back up web logging on our existing incubator. Ian wants to help with this.

Lasse has a contact at Lego that will supply NXT and other parts for any Lego-based equipment we design. Lasse would like assistance designing a compact machine to photograph plates on-demand via Internet services.

Lasse took some photos - we’ll post those once they’re online.

As always, please speak up if you want to work on any of our projects!

General Meetup Oct 6

Fri, 30 Sep 2011 10:29:36 -0400

General meet up & interest meeting
Thursday October 6, 2011 at 8:00 PM
At Sprout (339R Summer St. Somerville, MA 02144)
Coincides with Sprout’s open project night

Project Status Meeting & IPA Bottling Oct 2

Fri, 30 Sep 2011 10:13:00 -0400

IPA Bottling & Project Status
Sunday October 2, 2011 at 2:00 PM
At Sprout (339R Summer St. Somerville, MA 02144)
IPA bottling and meet up. Will get status on electrodeposition project and establish leads for the webcam and transformation projects.

IPA Bottling & Meetup

Wed, 21 Sep 2011 10:03:25 -0400

Friday September 23, 2011 at 8:00 PM
At Sprout (339R Summer St. Somerville, MA 02144)
IPA bottling and meet up. Will get status on electrodeposition project and establish leads for other projects.

Does Vibrio fischeri bioluminescence follow a circadian rhythm? Circadian, no. Rhythm, yes.

Wed, 13 Jul 2011 00:29:12 -0400

Does Vibrio fischeri bioluminescence follow a circadian rhythm? Circadian, no. Rhythm, yes.

Culturing bioluminescent microbes from squid

Mon, 11 Jul 2011 01:48:27 -0400

Incubated a fresh squid (from haymarket friday night) in a plastic bag for 2 days in a refrigerator. Does it glow?

answer: not yet. Smells, though.

Transferring it to a tupperware container cracked to provide some airflow.

Incubating with a cover on the benchtop.

fuck yeah ethics

Wed, 29 Jun 2011 18:33:18 -0400

fuck yeah ethics

Don’t see any colonies from the replated lyocomp 116 +...

Wed, 29 Jun 2011 18:31:00 -0400

Don’t see any colonies from the replated lyocomp 116 + bba_j04450 :(

try starting over by growing from the agar stab we made back in may? (stabs are in the “stabs” box in the 4C fridge)

For today: Make stronger IPTG plates and streak them. See...

Sat, 18 Jun 2011 00:00:00 -0400

For today: Make stronger IPTG plates and streak them. See evernote lab notebook for details.

2011-06-12 J04450 in lyocomp 116 on AMP + IPTG plates

Tue, 14 Jun 2011 17:01:00 -0400

The plan:

pipetted & spread 100 uL of 0.1 mM IPTG onto AMP plates, let dry.

Picked a J04450 colony and streaked it on two plates 30 min later. Looking for induction of the RFP in J04450 via IPTG repression of the LacI repressor.

24 hours later, no visible induction :(

Note, spilled ~3 mL of 1M IPTG after sterilizing it. MSDS had no clear clean-up information. Wiped up the spill w/ paper towel and put it w/ autoclave waste. IPTG is heat-sensitive. Washed the table 3x with water and then with bleach.

It would be excellent to have more operable “scram” instructions for situations like this (exactly what to do if a spill or exposure) before working with chemicals. Would like to develop these every time a new chemical is ordered, before it arrives at the lab. Not sure what the best way is to do.

Transformation results 2011-05-16

Tue, 17 May 2011 15:31:31 -0400

Went over to the bosslab yesterday (16 May 2011) around 11 PM to check on the plates (18+ hrs of incubation in the “incubator”).

No signs of colonies. Transformation failed, or amp was too conc, or incubator was too hot, or…

SOC broth that was incubated was mildly turbid. The biobrick card used was not sterile, so the organisms were probably not transformed E. coli. Plated 100 uL of the SOC on LB agar plates anyway. We’ll see.

lab notes in evernote

Logging incubator temp w/ pachube, arduino, & DS18B20 sensor

Tue, 17 May 2011 14:38:00 -0400

The “incubator” we’re using at the bosslab is really an oven. Instead of temperature gradations on its control knob, it just has 5 numbers, “1-5”. With some trial and error we’ve got it set to a temperature between 28 C and 40 C.

But I wanted to see how responsive the temperature control system in the ~~oven~~ incubator is, so I set up a simple data-logger to record the temperatures inside it.

A Dallas Instruments temperature sensor (DS18B20) is hooked up to an arduino, which sends the temperature measurement once a second to a computer via “serial”. A processing sketch on the computer uploads the data to Pachube.

Here’re two dynamic graphs of the temperature data stream from pachube. (a dotted line indicates a data failure; probably because the computer was shut off, fell of the network, processing stalled… etc).

As you can see, the values oscillate on a roughly hourly cycle over a range of about 10 degrees C. I wonder how that affects the growth of E. coli… hmmm.

BOSSLAB 2011-05-15

Sun, 15 May 2011 23:05:00 -0400

Goal: transformation of Invivogen lyocomp cells w/ BBa_J04450 on card (circa 2008).

We’ll be back in 18 hours w/ photos of the results. Here’s what we did today:

2011-05-11 notebook

Thu, 12 May 2011 15:40:00 -0400

Preflight

what’s our goal

dry-run through transformation protocol w/ BBa_J04450 on card using Invivogen lyocomp cells

lyocomp 116 cells, e. coli k12 (?), non-pathogenic
hot water bath; 42C, heat caution
Ian: “The chemicals we’ll be using amount to waste water (E. coli), cheese whey (tryptone), Vegemite (yeast extract & NaCl), tofu firming salts (MgCl2 and MgSO4), jelly (agar) and antibiotics and the concentrations are fairly low, so no special dangers - i.e. don’t eat it. Cleanup can be city sewer, since all of these are things that are already found in waste water.”

risks to us

trivial; just doing a dry run w/ the water baths
conventional fire risk w/ propane burner for autoclave - use it outside on asphalt

risks to community

fire hazard from propane burner

what waste are we gonna generate

hot water
combusted propane gas

Protocol

Thaw competent cells (Lyocomp E. coli) on ice.
During this time pre-chill sterile centrifuge tubes on ice.
Gently tap competent cell containers to mix
Aliquot bacterial suspension in pre-chilled tubes (100 µl per tube).
To the aliquots add 1 µl of plasmid (RFP PUC19).
Gently tap centrifuge tubes to mix
Incubate 30 min on ice.
During this time pre-heat water bath to 42°C (don’t use heating block).
Incubate samples at 42°C in the water bath for 60s. Do not shake.
Immediately chill samples on ice for 2 minutes.
Add 0.95 ml of room temperature SOC.
Plate 10 µl of bacterial suspension on antibiotic-containing agar plate. (filter sterilized Kanamycin100 ug/ml?).
Transfer some bacteria to another antibiotic-containing plate using a loop.
Incubate plates at 37°C for 18 h.

visual protocol

PCR Cleanup

Tue, 27 Apr 2010 01:24:00 -0400

Earlier today I emailed all of the participants in the recent Reading Genes workshop. Here is what I said:

Thanks so much for participating this weekend in the diy-genotyping workshop. I personally had a great time working with all of you and I learned a wonderful amount preparing and running the event. It was a bit of an experiment, and if some or all of the results are negative, rest assured that I will figure out what went wrong and invite you back for a successful second try.

Today I’m purifying everyone’s PCR products for sequencing. I’m using an enzyme I bought from Affymetrix (the ExoSAP-IT kit) to do the purification. The sequencing machine wants 12 uL (micro-liters) of each person’s purified PCR Product. So, I’ll take 12 uL of unpurified PCR mix from each person’s tube, add it to a new tube, and also add 4.5 uL of the magical ExoSAP-IT mix.

Theoretically, the included enzyme will chew up any remaining non-PCR-product DNA and remove their phosphate backbone. The genomic DNA we copied, the primers, and the DNA nucleotide building blocks that we built the copy from (the As, Gs, Cs, and Ts) will be changed from being nucleoTIDES to nucleoSIDES. I can’t give the sequencer tubes with active ExoSAP-IT, since it would probably mess up the sequencing reactions (we’re using “Sanger sequencing”, which is like a specialized kind of PCR + gel electrophoreses), so I’ll denature the ExoSAP-IT after it does its work by heating the entire mix to 80 C for 15 minutes.

So that’s the plan. I’ll let you know how the sequencing goes.