July 27, 2009 - 10:42am
What is general transduction rate ( in a bacteria using any phage?)
I am doing trasductaion in Ecoli using p1 vir , using Ecoli gnome manipulation by p1 transducation.
when I plate the tranduced cell on Km- EGTA plates i hardly see 1 or 2 colinies. So I was wondering what would be the number on colonies we see is a good transuction?
February 16, 2010 - 12:29pm
#2
Tnx
Thanks CP expert for your nice explanation
actually, I can't get any colonies after transduction?! :( (I'm looking for kan resistant colonies after transduction)
February 18, 2010 - 11:23am
#3
generalized transduction
Did you check out the titer of the P1 stock you use? A good P1 stock titer is 109 to 1010 pfu/mL. Since your transduction is problematic, you should titer your phage stock or use a fresh one. If the titer is too low then you will not be able to isolate tranduced colonies.
April 8, 2010 - 12:46pm
#4
obtaining fresh P1
Where can I get fresh P1? I can't seem to find it for sale anywhere.
April 9, 2010 - 8:37am
#5
P1 stock
Unfortunately P1 stocks are not commercially available. Usually people hand them around from person to person. The best would be to contact a phage lab in your department and get a hold of high-titer P1.
You can always titer your own stock. If you find that the titer is low (<109 pfu/mL) then make a new lysate. You can find protocols for both in Unit 1.17 of Current Protocols in Molecular Biology on “E. coli genome manipulation by P1 transduction (Thomason et al (2007). Hope this helps!
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Transduction refers to the transfer of bacterial DNA from one bacterial cell to another by means of a virus particle that infects bacteria, i.e. a bacteriophage. Generalized transduction occurs when bacterial DNA (instead of phage DNA) gets packaged into newly-forming phage heads by error. The error rate is about one phage particle in 1,000 that contains bacterial instead of viral DNA. This process is termed “generalized” because the bacterial DNA packaged in the phage head can be derived from practically any part of the bacterial genome. The phage containing bacterial DNA is then used to infect a second bacterial cell and thus generate a transductant. Recombination of the injected DNA happens ~10% of the time. Assuming e.g. that 2% of the bacterial chromosome fits into the phage head, the chance that transduction will occur with any given bacterial gene is ~(1/1000)(1/50)(1/10)=2x10-6. Rates of generalized transduction for a specific gene range from 10-8 to 10-5.
Have a look at a recent review on generalized transduction by Thierauf et al (Methods Mol. Biol. (2009) 501, 267-286), as well as at Unit 1.17 of Current Protocols in Molecular Biology on “E. coli genome manipulation by P1 transduction (Thomason et al (2007) Curr. Protoc. Mol. Biol. ;Chapter 1:Unit 1.17). Both contain detailed protocols on generating transductants using P1 bacteriophage as well as sections on troubleshooting that will help you out optimize your protocol.