The GFP Project: One Step Backward, Two Steps Forward

Oops.  After a week off for spring break, I returned to the GFP project and realized we had to do another transformation, because last time we had accidentally used up all our stocks of transformed bacteria.

So we did another transformation, and we also plated some of the leftover bacteria we had on a plate from the first transformation.  Hopefully we can use one of these sources to grow an overnight culture and extract GFP from.

We also had some interesting visitors!  We had Jonathan, an anthropologist in the science and technology field, who knew Mac Cowell from way back, and that we had some DIYBIO screens hidden away at BOSSLAB!  Time to make some T-Shirts!

We also had Kris Constable and Megan who are starting BioSpace, a which is exactly what it sounds like (a bio-hackerspace) up in Canada.  It was cool to meet international DIYBIO folks!

The other good news is that the stickers are finally in the envelopes and addressed!  They should go out this week.

The GFP Project Week 1: A Lesson in Patience and Ingenuity

Arduino: the alternative lighting platform for molecular biology

It had been two days since the transformation yesterday, and I had not seen any growth on my plates.  I was becoming concerned.  It can be tricky to orchestrate the teaching and the actual transformation procedure, and I have had less than optimal results with this media, strain, and plasmid before.  I was not sure what I would do on Sunday if there were no transformants!  It would be a disaster, and very demoralizing.

I had a meeting to go to that would result on me being on the red line, so on the way back I stopped by and took a look at the cultures with my blue LED.  Nothing was growing, except for what appeared to be some e. coli growing on the ampicillin plate, which I took to be an ill omen.  I left my blue LED and some batteries there, because I figured that if there were transformants, they would be more likely to grow on the multitude of plates at sprout instead of the two that I had.

I wasn’t sure what to do there, so I grabbed a few items for doing another transformation and headed home, convinced that I would have to do another transformation.  Upon my return home, I obsessively checked again.

Perfect little colonies!

When I returned from dinner, I decided to take one last look.  I was greeted with two plates full of transformants, in perfect little green colonies.  Having left my sole blue LED at sprout, I initially thought that I would have no way of testing the fluorescence, until I spied my arduino nano on my desk.  I remembered that it had a blue LED on it, so I plugged it into my laptop and used it to light the plates, which indeed fluoresced.

BAM. GFP!

Lessons learned:  Be patient.  Synch your life to the organism you are studying, not the other way around.  Also arduinos are good for many things.

cell growth phase chart, found via the google

The final question you may have is “why did they take so long to grow?”.  The answer there is something I should have recognized!  I totally forgot that there is often a “lag phase” of growth in bacteria.  This is the phase where bacteria are generating the needed metabolites and substrates to adjust to their surroundings.  The colonies are also growing (in this case) from a single bacteria!  So it makes sense that there was a pretty big lag from when they were plated, until  I could see them.

The GFP Project Week 1: Hands-on Transformation!

The shirt says it all.

The GFP project rolls forward!  Powered (funded) by the people doing the work, I managed to order all the materials that we would need for a transformation this weekend (by that, I mean today).  There was an awesome turnout; some new people came out, a lot of supporters came out, and a good time was had by all.

From left to right: LB broth, LB agar, 50 mM CaCl solution, alcohol burner, tape, streaked plate, arduino, pipettes in a fancy box, ziplock-o-wires

To start out, we had to sterilize some media and CaCl2 solution.  We made 200 ml of LB agar, 80 ml of LB broth, and 500 ml of 50mM CaCl2 salt solution.  The LB agar was used to make LB+ ampicillin plates.  These were prepared by adding 2 ml of 1% ampicillin to the still-molten agar once it had cooled to “warm enough to hold”, and then pouring it into sterile petri dishes.

The Calcium Chloride solution and the LB broth were needed for the transformation.  You only need 250 ul of each per-transformation.  We prepared the LB broth in bulk because it is useful to have on hand, and we prepared way to much CaCl2 because our scale was not sensitive enough to measure out a smaller quantity that would make a 50mM solution.  We could have made a 1M solution and diluted it, but it seemed simpler to just make 500ml.

Totally legit ice-bucket of science

The other necessities for the transformation that had to be prepared were the ice bath, and the 42 C water bath.  The ice bath was created using the insulated shipping crate that the plasmid and ampicillin came in by throwing some ice in the larger (bottom) container.  The water bath I have successfully made before by nuking (microwaving) the water and then  guesstimating that it was hot enough (more than body temperature, but cool enough to hold, between 37-50C).  Today I used what seemed to be a much more reliable method, which was a hot plate and a submersible temperature probe.

(Hot) Water bath setup. Sensor in falcon tube taped to beaker, arduino connected to sensor and computer

The temperature probe was improvised with my arduino mini, a DS1820 digital temperature sensor, some long wires, and a 15 ml falcon tube.  I submerged the falcon tube in the water bath, and stuck the temperature sensor (which had been soldered to the long wires) into the tube, allowing it to (hopefully) measure the temperature of the bath.  This was reporting the temperature back to my computer with some script I got to work a while ago.  A better way to set this up physiclly would be to cram the sensor in to a thin-wall reaction tube (PCR tube) and fill it with milliQ water (non conductive, very DI RO water), stick the sensor in the water and waterproof all that with sugru or epoxy.  This way there would be no air gap, and the sensor would be safe in DI water.

The game plan for the transformation in flowchart doodle format

During all of that setup we were also preparing the bacteria for heat shock, teaching people how to pipette and streak bacteria, and talking about biology in general.  Our transformation protocol, officially was this:

Gather all the things! Everything didn’t fit in this picture, but this is a lot of stuff!

1.  Gather reagents (LB broth, 50mM CaCl2, LB+Amp plates, e. coli plate, eppendorf tubes), prepare ice bath and water bath, and gather your tools (flame, inoculation loop, pipettes and tips)

Use your sweet pipetting skills to add salt solution to your eppendorf. keep on ice.

2.  add 250 ul of 50 mM CaCl2 to an eppendorf tube, and chill on ice

Scrape e. coli off your source plate. Be careful not to pick up any agar!

3.  scrape off some e. coli (enough to see on the loop) with a flame sterilized loop, and swish the loop around in the iced CaCl2 in the eppendorf.  Make sure they fall off into the CaCl2.

flick! flick! done!

4.  flick the tube until the bacteria are no longer clumped together.  The solution should be cloudy now.  Chill tube on ice for 1-5 min.

Adding tiny amounts of DNA means tiny pipette tips!

5. Add 20ul of plasmid DNA at .005ug/ul to the eppendorf.  This is .1ug of DNA.  Return tube to ice, incubate for 5 min

Heat shock!

6. heat shock at 42C for 90 seconds

7. add 250ul of LB broth

Spread the bacteria around with the sterile loop

8. plate 100ul on LB+Amp plates.  Pipette 100ul on to the plate, then flame loop and cool in agar somewhere that you did not pipette onto.  Use loop to spread pipetted transformant mixture.

With all of that done, we cleaned up and headed off to wherever.  Some people had to leave early, but most everyone seemed like they would be back!  There should be results on if this worked in just a few days.

The GFP Project Week 0: Welcome to BOSSLAB

Something is cooking at BOSSLAB!

The GFP project has begun at BOSSLAB.  Headed by the Chief Troublemaker of this blog (me), a group of people will be using BOSSLAB to play around with pGREEN, a plasmid that produces GFP.

The goal of this project is not only to get local people working hands-on with biotech, but to support the DIYBIO community by producing a model project, with documentation, that people can do.  Over the next month or so I will be cranking out all the information you need to safely create and modify genetically engineered organisms in an environment about as sterile and complicated as your kitchen.

The GFP project will have # components, as listed  below:

  • Transform pGREEN intoe. Coli MM294
  • Extract pGREEN from the transformants and verify the quality of the plasmid DNA
  • Extract GFP from the transformants for extra points
  • ????

This week was kind of like “Week 0” for the project in that I showed everyone how to pour plates, streak for individual colonies, and practice (some amount) of aseptic technique.  As I mentioned, one of the goals for me in leading this project is to show people DIYBIO to show people basic techniques.  To make sure I do a good job, I will be covering the techniques we use in the project in detail in separate posts.

Hopefully we will get to do the first item next weekend, at the twice-montly BOSSLAB meeting.  It will be nice to have something really going on during the meeting; maybe more people will get interested and join the project, allowing us to get some nicer things for the project.

After this project comes ????.  That means I don’t know if we will continue doing molecular biology, or switch to something like microbial diversity, or try to do some kind of Mendelian genetics project with plants.  There are a lot of really cool biological things to investigate, and although molecular biology and synthetic biology are my favorites, BOSSLAB is really open to anything.  If we continue in the molecular biology direction, we could try doing a restriction digest on the extracted plasmid and run it in a gel, or have part of it sequenced (find out what kind of GFP we are making!), or ligate it to a localization tag and see it expressed in only part of the cell (contingient on getting our fluorescence scope working).  Really, there are all kinds of possibilities for this!  But first, extraction and verification have to happen.