We're collaborating with some physicists on a cool laser-tweezers experiment. The tests are done on single cells, so it's important that the cells used for this experiment are as competent as possible. Our collaborator just asked " Is there some quantitative way to go between the transformation frequency of a culture and the fraction of cells that are competent, so we'll know, for a particular batch of cells, how many we need to try?
Yes, and it's a very simple old-fashioned experiment.
Cells that are maximally competent take up at least several DNA fragments. If we give them chromosomal DNA carrying two different antibiotic resistance genes (say novR and nalR), some cells will become resistant to both antibiotics because they took up both genes (double transformants). For this analysis it's important that the two genes be far enough apart on the chromosome that they will be in different DNA fragments. If all the cells are competent, we can predict the frequency of these double transformants; it's the product of the frequencies of single transformants. So if 1% (0.01 or 10^-2) of the cells in this experiment became NovR, and 0.5% (0.005 or 5x10^-3) of the cells became NalR, we predict that 0.005% (0.00005 or 5x10^-5) of the cells will be NovR NalR.
But what if not all the cells were competent? Say only 10% of them were. Then the 1% transformation frequency to NovR was because 10% of the cells had a transformation frequency of 10% and 90% had a transformation frequency of 0. Similarly the 0.5% transformation frequency to NalR would be because 10% of the cells had a transformation frequency of 5% and 90% of the cells had a transformation frequency of 0. Now the predicted frequency of double transformants is different. The 10% that are competent will have a double transformation frequency of 0.5%, and the 90% not competent a frequency of 0, giving a total frequency of 0.05%. This is ten-fold higher than if all the cells were competent.
So, a double transformant frequency higher than expected if all the cells were competent means that only a fraction of the cells were competent. On the other hand, if all cells were only a bit competent, so most cells were only taking up a single fragment, the same analysis would give a double transformation frequency lower than expected. This was all worked out 30 or more years ago.
When we do this analysis on competent H. influenzae cells, we always find that the double transformant frequency is higher than expected if all the cells were competent, which means that only some of the cells are competent, and that the competent cells are taking up multiple DNA fragments. (For reasons having to do with how the DNA is integrated into the chromosome, there's a fudge factor of 2.) When the transformation frequency for the single markers is high, the calculations indicate that about half of the cells are taking up DNA. When the single-transformant frequency is low (in some mutants, and in cells from conditions that only weakly induce competence), fewer cells are competent, but the ones that are are still taking up multiple DNA fragments.
So, we've usually used the single-marker transformation frequency as an indicator of the fraction of the cells that are competent. Maybe for the laser-tweezers experiments we should check again.
- Home
- Angry by Choice
- Catalogue of Organisms
- Chinleana
- Doc Madhattan
- Games with Words
- Genomics, Medicine, and Pseudoscience
- History of Geology
- Moss Plants and More
- Pleiotropy
- Plektix
- RRResearch
- Skeptic Wonder
- The Culture of Chemistry
- The Curious Wavefunction
- The Phytophactor
- The View from a Microbiologist
- Variety of Life
Field of Science
-
-
Don't tell me they found Tyrannosaurus rex meat again!2 weeks ago in Genomics, Medicine, and Pseudoscience
-
-
-
Course Corrections4 months ago in Angry by Choice
-
-
The Site is Dead, Long Live the Site2 years ago in Catalogue of Organisms
-
The Site is Dead, Long Live the Site2 years ago in Variety of Life
-
Does mathematics carry human biases?4 years ago in PLEKTIX
-
-
-
-
A New Placodont from the Late Triassic of China5 years ago in Chinleana
-
Posted: July 22, 2018 at 03:03PM6 years ago in Field Notes
-
Bryophyte Herbarium Survey7 years ago in Moss Plants and More
-
Harnessing innate immunity to cure HIV8 years ago in Rule of 6ix
-
WE MOVED!8 years ago in Games with Words
-
-
-
-
post doc job opportunity on ribosome biochemistry!9 years ago in Protein Evolution and Other Musings
-
Growing the kidney: re-blogged from Science Bitez9 years ago in The View from a Microbiologist
-
Blogging Microbes- Communicating Microbiology to Netizens10 years ago in Memoirs of a Defective Brain
-
-
-
The Lure of the Obscure? Guest Post by Frank Stahl12 years ago in Sex, Genes & Evolution
-
-
Lab Rat Moving House13 years ago in Life of a Lab Rat
-
Goodbye FoS, thanks for all the laughs13 years ago in Disease Prone
-
-
Slideshow of NASA's Stardust-NExT Mission Comet Tempel 1 Flyby13 years ago in The Large Picture Blog
-
in The Biology Files
Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
3 comments:
Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS
Subscribe to:
Post Comments (Atom)
Could you explain the fudge factor of two a little bit? I'm interested to know how/why/if it is exactly two.
ReplyDeleteI think it's somewhere between 0 and 4. It arises partly because only a single strand is replaced. This ccreaters a two-fold dependence of TF on whether the cells divide before being plated. The strand that's recombined in may also be 'corrected' by mismatch repair.
ReplyDeleteI think it's somewhere between 0 and 4. It arises partly because only a single strand is replaced. This ccreaters a two-fold dependence of TF on whether the cells divide before being plated. The strand that's recombined in may also be 'corrected' by mismatch repair.
ReplyDelete