Periplasm preliminary experiments

This post explain a series of preliminary experiments that have as an objective to answer three specific questions about  a periplasmic DNA extraction procedure that has been described by Kahn et al 1983, Barouki & Smith 1985, and Mell et al 2012.

Questions to be asked are:

1. Is there any contamination of DNA fragments not taken up during the washes steps (step 3 figure below)

2. Is there any chromosomal DNA contamination in the periplasmic extraction?

3. How much DNA is been extracted by the periplamic DNA extraction

The protocol is based on a DNA uptake essay that will be done in Haemophilus influenzae a naturally transformable bacteria followed by a organic periplasmic extraction using Tris 10mM EDTA 10mM, CsCl and phenol:acetone. The periplasmic extract will be used as donor DNA in a transformation essay following Poye & Redfield (2003) protocol. This transfomation should be sensitive enough to detect DNA present in the periplasm.

Overview of protocol described in figure below

mm1

The first question will be answered by using  a Rec2(ComEC) knockout and a pilA knockout as recipient cells and sheared genomic DNA (average 6kb bp) of Map7 (an Rd strain with seven different antibiotic resistance markers). Rec2 is inner-membrane porin that is involved in DNA internalisation and knockout mutants can take up DNA but are not able to translocate DNA to the citoplasm. On the contrary, PilA knockout mutants are not able to take up DNA.

A. ) If the washes at step 3 worked effectively then I should expect to see no colonies in the transformation (step 9) using pilA knockout cells and plenty using Rec2 knockout cells

B.) If I see colonies in pilA mutant, I am going to have to re-think how to remove not-taken up DNA. I might increase washes.

The second question will be solved by using Kw20 cells with a nov or kan antibiotic marker on their genome as recipient cells and using sheared genomic DNA of NP with Nal antibiotic marker. By having two antibiotic markers I can assess the amount of chromosomal DNA that leaks to the periplasmic extraction and compared it to the amount of DNA recovered from the periplasm.

This brings me to  my third question, how can I quantify the amount of DNA extracted in the periplasm. This relates to my second question as based on the colonies that I get in the transformation I could estimate the donor DNA amount but I need to make a calibration curve assessing the transformation frequency at lower amount of donor DNA (from 1 ng to 20 ng; 5 or 6 points taken).

Another alternative is using a fluorometric dye such as Qubit reagents. There is no much information about the dye used in Qubit essays but it seems according to the manual that it bind specifically to dsDNA as shown by the figure below and it is much more reliable than nanodrop quantification. What I am not sure is that there is no information about how often it binds to DNA (every 10bp, 20bp, 100bp?). I know that some real time PCR essays use saturating dyes that bind a very often to DNA molecules but I was not able to get that information (and given the secrecy of some kit providers I might not able to find it)

qubit

 

 

 

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One thought on “Periplasm preliminary experiments

  1. Rosie Redfield

    Some quantitative questions:
    For the pilA mutant, is one colony unacceptable? From how much of your recovered DNA?
    For the rec2 mutant, how much DNA do you need to recover? How many colonies would this give?
    How much chromosomal DNA contamination would be too much?
    For the possible Qubit assay: How does the assay’s sensitivity compare with the amount of DNA you need to recover? What about the sensitivity of the transformation assay?

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