OpenTrons E. Coli Transformation (Part 1)
OpenTrons Transformation Success!
We have completed our first set of automated E. coli transformations on the OT.One robot! Thats right, we got a robot to insert DNA into a bacteria that makes it pink.
What is a 'Heat Shock' Transformation?
A 'heat shock' is when you change the temperature of the cells quickly -- heat them up! -- so that new DNA flows from floating in solution around the cell, through its membrane, and into the inside of the cell. Then, hopefully, the cell will incorporate the new DNA into its genome and express the characteristics it codes for.
Heat Shock Hacking
In the typical heat shock transformations done in labs every day, tubes containing cells and DNA are moved by hand from a cold water bath to a hot water bath, and then back again. However, water baths and electronics dont mix well, and tube grippers are really expensive. We are all about providing low-cost automation to hundreds-of-thousands of labs still doing everything by hand, so we had to hack the heat shock.
We start with two tubes (rather than one), each already in either the ColdDeck or HeatDeck. Then, the robot simply transfers the cell + DNA solution from the tube on the ColdDeck to the tube on the HeatDeck, and back again (instead of moving the tubes themselves). This achieves the same transformational effects of the manual process, but is 100% automated!
1 - Chill the cells at 4 degrees Celsius on the OT-ColdDeck to make their cellular membrane rigid.
2 - Move the cells into a new tube sitting on the OT-HeatDeck at 42 degrees Celsius for 60 seconds -- the heat shock! This causes the cells to expand very quickly, creating holes in their membrane for the DNA to flow into.
3 - Move the cells back to the OT-ColdDeck so they cool down to 4 degrees Celsius again, closing the cell membrane holes and trapping the new DNA inside.
4 - Add LB broth to the cells and wait for 10 - 30 minutes. Then plate the cells by hand and put in the incubator.
Both transformations we've done so far were successful! As you can see in the photos, there are a large number of colonies, all expressing the RFP. Both plasmids also contained a selective marker for antibiotic resistance (ampicillin for try 1, chloramphenicol for try 2).
Competent Cells: Invitrogen 'Top 10' K12 E. Coli.
Plasmid 1: pSB1A3 backbone with ampicillin resistance and RFP gene insert (part BBa_J04450 from the iGEM registry). Plated on LB with ampicillin.
Plasmid 2: pSB1C3 backbone with chloramphenicol resistance and RFP gene insert (part BBa_J04450 from the iGEM registry). Plated on LB with chloramphenicol.
New Robot Modules
This experiment would not be possible without our new ColdDeck and HeatDeck modules. The cold deck holds a temperature of 4 degrees Celsius, and the heat deck can go up to 70 degrees Celsius. Stay tuned, will begin selling these add-ons in the coming weeks!
Now that our proof of concept tests are complete, it is time to flesh out our results!
More data is always better, and our plan is run several more transformations in the near future. In this next round of testing, we will be scaling up to include multiple samples and controls per run, and calculating the transformation efficiency of our protocol.
Special thanks to Will Shindel from Genspace for working with us on this!