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.
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.
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.
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.
4 thoughts on “The GFP Project Week 1: A Lesson in Patience and Ingenuity”
Had a similar issue when teaching students in an academic environment once, and it highlighted to me one of the big issues when dealing with transformed or conjugated cells:
When cells form a “colony”, we say it was formed from a “colony forming unit”, rather than a cell, because often you have clumps of identical cells that land in one place, forming a colony.
However, in this clump of cells, only one may become transformed or successfully conjugate, and so effectively the size of the “colony forming unit” drops from 1-20 to 1. With a doubling time of 20 minutes (the ideal time for rapidly growing lab-strain E.coli), the difference in time taken for 1 cell to grow to a visible colony, and clump of 16 cells to grow to a visible colony is 1 hour and 20 minutes.
In the class I was teaching, their “before conjugation” plates all had teeny tiny pinhead-sized colonies all over, the “after” plates seemed empty. We were expecting plenty of trans-conjugant bacteria, but with such small “before” colonies, the “after” bacteria were probably still too small to see! If it had been my experiment, I’d have put them back in the incubator, but sadly we couldn’t.
Patience: A critical character trait in biology.
I really appreciate this blog. It has been immensely helpful to me.
I was wondering what your experience with a DIY transilluminator might be? I’m working on a slightly intense project, using Processing, Arduino, and some sensing equipment to make a specialized transilluminator. Thanks again for the blog. I’m enjoying reading it.
I think my favorite transluminator/dye for DNA is gelgreen+blue LEDS. It lets you not use UV, which is convenient. What are you working on?
Glad you are enjoying the blog,
Thanks for the reply. The blog is great indeed. I’m actually doing Bacterial transformations… Using eGFP and (hopefully if it goes well) T-Sapphire. I’m worried about the luminescence under just above UV light. The eGFP should be visible at around 400nm, however the T-Sapphire…Maybe not. PErhaps I will have to go with two sets of LED’s which makes things a little tight on space. The LED arrays are tiny (1.5cm in diameter).
I build an environment that turns human presence into LED activity allowing for the eGFP to be excited with interaction.
Anyway, keep up the good work. I appreciate it very much!