Transformation 2.0- RISD

A few months ago, I was invited by Sara Wylie to talk to some students at RISD about DIY-Bio, specifically, I was invited to show them how I did my DIY transformation a few months ago.  It was an AWESOME experience, and a lot of interesting topics came up in the area of ethics, and most importantly who should be allowed to practice or regulate biology and biotechnology, which are controversial and important.  And too big for this post, which will be about another exciting transformation!

We used the same procedure, as described before in my transformation post.  I wasn’t around in RI for the next week to check on the plates, but Sara sent me some pictures.  It is interesting that this transformation has the same problems as the previous transformation.

Transformants?

Again, there are what look like contaminants/giant satellite colonies.  Again, the GFP expression is very subtle.  I am curious about what the non-green colonies could be.  Maybe the incubation to express the ampicillin resistance is too long, and the b-galactosidase is breaking down the ampicillin when the cells are plated.  I used the same MM294 strain of e. coli and the same pGREEN plasmid as I did last time, but both were ordered fresh from Carolina Biological.  If I don’t see anything that would explain this in my research on the MM294 and satellite colonies, I will probably try this with a different strain.  Maybe that will be a BOSSLAB project for next year (if you are near Boston, and want in, send me a message!).  Anyways, here is a picture of the same plate under a blue LED.

Transformants expressing GFP, which is fluorescing under a blue LED

Another protip I have from this, and from yesterdays experiment (TSS transformation) is that you should pour your plates way before you intend to spread on them.  This lets them dry out a little bit before you add a bunch of liquid do them.  You can even put them in an incubator to dry them out a little!  We had some difficulty getting the plates at RISD to absorb 100ul of transformants in broth.  Yesterday a plate I had easily absorbed 300ul of transformants and broth, which was poured the day before, and then pre dried in an incubator for 10-15 minutes.  Just keep an eye on them and don’t let them dry out!  By drying them you can also avoid the ring of growth around the outside as seen below.  This seems to be because bacteria get in condensation/broth/wetness on the plate, and then roll around on the plate.  Surface tension keeps it stuck between the plastic wall and the agar, creating the ring.

The ring of BAD. Note growth around the edges of the plate.

Eeew!  So keep your plates dry.  But not too dry.

These bacteria were eventually used in one of the final class projects, here.  There is a pretty swanky picture of a bacto-QR code on that page, definitely check it out.

Coming soon: Dinoflagellates and MORE transformations.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s