Monthly Archives: January 2016

Exposure vs Dose

My supervisor asked to calculate my radioactive exposure, after working with P32, for our annual radioactivity report. My first thought was that don’t remember very well how to do that calculation, so I went back to the radioactivity manual I got during my training. I looked for a formula to calculate exposure and I see that the manual says:

The theoretical dose to an individual in the vicinity of a point source of radioactivity is defined as:

 

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Perfect! Now I only need to The specific Gama Ray Constant. Wait a minute!! Does P32 emits gamma rays? The manual gives me a list of isotopes and their gamma ray constants but P32 is not in the list!

How am I supposed to calculate my exposure then?

Ok so after hours of reading the manual carefully I decided to look for answers at the place where most people would start looking… Wikipedia

Well, it turns out P32 does not emit gamma rays. Perfect, that is a start, but HOW I CALCULATE MY EXPOSURE THEN?

Wait! the manual uses the terms exposure and dose, but I do not understand the difference (At least by reading the manual). So, I decided to look for other manuals.

After several more hours, I found the best manual explaining the concept between exposure and dose. So, “Dose is a measure of energy deposited by radiation in a material, or of the relative biological damage produced by that amount of energy given the nature of the radiation”, while “Exposure is a measure of the ionizations produced in air by x-ray or gamma radiation. The term exposure (with its ‘normal’ definition) is sometimes used to mean dose. (e.g. ‘He received a radiation exposure to his hand.’)”

Ahhh! that is why I was confused, people use “exposure” to mean “dose”.  So, I do not have to calculate my exposure but my dose to P32.

Now, how can I calculate my dose?

Most of the radiation dose calculated wil be for the skin (hands) since my body was protected.

Looking at the Radionuclide Safety Data Sheet of P32 I realize that the Dose Rate by P32 is 348 rad/hour per 1 mCi/cm

Rad is a unit of absorbed dose

Rem is a unit of dose equivalent (since dose of alpha particles is not the same as beta or gamma)

For beta emitter 1 Rad = 1 Rem

Ok now I can use the formula above. I do not have a gamma ray constant but I have the P32  Dose Rate which will replace the T in the formula. The average activity (A) used per experiment was 0.74 MBq. The exposure time (T) lets say is 10 hour (probably an overestimation). The distance (d) from the source was 30 cm2 approximately.

((348 rem/hour per 1 mCi/cm) * 0.02 mCi * 10 hours)/ (20cm) = 3.48  rem

3.48 rem to SV

1Sv/100rem * 3.48 rem/year =    0.0348 Sv/year = 34.8 mSv/year

 

Ok that dose is very low compared with the maximum dose per year of UBC nuclear energy workers and even general public.

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Lets consider the time I worked with the whole source with has 9.25MBq activity

Then time (t) would be marginal 0.25 hours and the activity (A) will increase to 0.25 mCi

((348 rem/hour per 1 mCi/cm) * 0.25 mCi * 0.25 hours)/ (20cm) =  1 rem

1Sv/100rem * 1 rem/year =    0.01 Sv/year = 10 mSv/year

So my total dose is 34.8 mSv/year + 10 mSv/year = 44.8 mSv/year

#update 31-01-2017

Note: This calculations are wrong. The radioactive safety commitee told me they will publish a guideline of how to do this calculation soon.

 

 

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Acinetobacter objectives

 

Coming back from holidays, my mind is still a bit slow in terms of thinking in perspective about my thesis project.  Last year, worked a lot in extracting sheared genomic DNA taken up by Haemophilus influenzae. As this part of the project is over, I have to start thinking in the DNA uptake process in Acinetobacter baylyi. This is not an easy process, maybe because my mind is still recovering from holidays or because I read most of papers about this topic several months ago.

In order to put my ideas in order lets start with objectives for the following weeks-months:

Main objectives:

  1.  Design and generate synthetic fragments with a degenerate region and illumina adaptors.
  2. incubate this synthetic fragments with Acinetobacter baylyi competent cells and extract periplasmic DNA (This DNA will be sequenced).

Objective 1 is under way. I have already designed the synthetic oligos which will help me generate the fragments and a former post-doc of the lab is helping check that my design is correct.

The second objective is a bit more complicated since before working directly in this, I need to work on some preliminary objectives.

Preliminary Objectives:

  1.  Test natural transformation protocol of Acinetobacter baylyi
  2.  Test self-specificity of Acinetobacter baylyi
  3. Test if the organic periplasmic extraction protocol works for Acinetobacter baylyi

Each of these objectives deserve a separate blog post, however, I will start with the first one.

  1.  Test natural transformation protocol of Acinetobacter baylyi

When I was working with Haemophilus influenzae I did not had to worry about getting a strain or about which markers I use to test natural transformation. Our lab had an extensive collection of strains with different markers to use. However, with Acinetobacter baylyi that is not the case. What do I have?  well…

Strains I have, include:

BD413 wild type

ADP239 comA::nptII

ADP239 comP::nptII

SDS-5

SDS-6

BD413 or ADP1 is a strain with high levels of natural transformation. It was derived from the soil isolate B4, and it has a small capsule which facilitate growing the bacteria in liquid media for easier centrifugation (Juni and Janik 1969).

ADP239 comA::nptII  is a mutant strain which has a nptII cassette (KmR) which insertionally inactivates comA gene (which is the Rec2/ComEC homolog in A. baylyi) (Friedrich et al. 2001). All ADP239 strains came from BD413, which have a mutation in the pobA gene. This mutation prevents bacteria the growth in mineral media when   p-hidroxybenzoate is the only carbon source (Porstendörfer et al. 1997).

ADP239 comP::nptII is a mutant strain which has a nptII cassette (KmR) which insertionally inactivates comP gene (Porstendörfer et al. 1997). ComP protein has similarities to the type IV type pilins and it has found to be essential for DNA binding as well as for natural transformation in A. baylyi (Porstendörfer et al. 1997).  This mutant will be used in my project as a DNA uptake negative control.

These first three strains were kindly donated by Dr. Beate Averhoff.

Strains SDS-5 and SDS-6 were obtained through the teaching microbiology laboratory. These strains are naturally transformable since each year they were naturally transformed by undergraduate students as part as teaching classes. SDS-5 is the wildtype strain, while SDS-6 is an auxotrophic mutant of SDS-5 that is not able to grow in the absence of tryptophan.

Which is the background of strains SDS-5 and 6, well I do not know. I still need to talk to the person that used to be in charge of the microbiology labs. However, several papers have used tryptophan auxotrophs for natural transformation (e.g. Juni 19721974Rokhbakhsh-Zamin et al. 2011). Based these papers mentioned early, SDS-5 and 6 could have derived from strains BD413 (Juni 19721974) or B4 (Rokhbakhsh-Zamin et al. 2011).

Problems or Complications

Natural transformation procedure in A. baylyi is apparently simple. The strain is cultured at 30 degrees overnight in LB. Then the culture is diluted 1 in 25ml in fresh mineral media and it is incubated for 2 hours more (Palmen et al 1993). Finally, the culture is incubated with DNA and it is plated in LB plates or minimal media agar with and without the appropriate antibiotic or carbon source (for minimal media).

Problems arise when mineral and minimal media are prepared. First, there is not a clear distinction between both media. I some papers, such as Palmen et al 1993 it seems that they are the same thing. Additionally, there are several variations of the media preparation and some are a long and complicated mixture of three solutions: a basal media, a trace element solution and a vitamin solution (i.e. Tschech and Pfenning 1984Porstendörfer et al. 1997).

The first thing that I did to start working in A. baylyi was streaking strains SDS-5 and SDS-6 in a minimal media that I made following the recipe of Palmer et al. 1993, which consisted in 60 mM lactic acid, 11 mM-KH2PO4, 95 mM-Na2HPO4, 0.81 mM-Mg SO4, 37 mM NH4Cl, 0.068 mM-CaCl2, and 1.8 uM-FeSO4. Since SDS-6 grows only in the presence of tryptophan, I expected that this strain would grow in LB agar, but not in the lactic minimal media agar; while SDS-5 strain should grow in both agar media. To my surprise, that was not the case, since both strains grew normally in both agar plates.

This result could be caused by three factors:

  1. There is contamination and the colonies on plates are not Acinetobacter
  2. There are traces of tryptophan in one of the elements of the media
  3. I froze the strains incorrectly

To discriminate between the three, I will:

  1. Look at strains at microscope to see if there is contamination
  2. Get the auxotroph strain SDS-6 again from the teaching lab, and streak it in LB and lactic minimal media agar.
  3. Streak SDS-5 and SDS-6 in LB, lactic minimal media and in minimal media prepared with the recipe from the teaching lab (which is different from the recipe I used).

Update (Jan 07 2016)

  1.  Colonies look normal, no contamination was observed
  2. I got another the Davis lab (they got the strain from the teaching lab) and the new SDS-6 grew on LB but not in the lactic minimal media (prepared by me). On the contrary, the SDS-6 that I got the first time grew on both LB and lactic minimal media.
  3. I also grew both strains in a minimal media plate prepared using the recipe from the teaching lab. Both SDS-6 and a SDS-5 grew poorly. Maybe more incubation time is needed

This results, on one hand, confirm that there is nothing wrong with the recipe I used to prepare the minimal media (which I got from Juni 1974, and Palmen et al 1993).  On the other hand, they confirm that the first strain SDS-6 that I got was probably mistaken by SDS-5.

A second problem that I must solve relates to figure out which marker I could use to test natural transformation. Three markers could be used:

  1. DNA from strains with nptII gene (KmR) incubated with Kanamycin sensitive strains
  2. DNA from wild type strain BD413 (pobA gene) incubated with ADP239 strains which are unable to grow in minimal media with p-hidroxybenzoate.
  3. DNA from SDS-5 or BD413 incubated with SDS-6 auxotroph mutant

Problems with this markers:

  1. I could extract DNA from ADP239 comA::nptII or ADP239 comP::nptII  and transform competent BD413 strain. However, this marker cannot be used to confirm the phenotype of the ADP239 comA::nptII or ADP239 comP::nptII strains since both have the nptII gene.
  2. Using pobA gene as a marker would be ideal, since this marker is the one used for natural transformation experiment done by the Averhoff group. However, I do not have the wild type ADP239 strain AAAGGRRR!!!!
  3. I could use SDS-5 DNA and select for growth in tryptophan but first, I must figure out what went wrong with the media, and second, knockout mutants of comP and comA are able to grow in tryptophan.

Solutions:

  1. It seems that the simplest solution would be to making my own ADP239 wild type. I could PCR the mutated pobA gene from ADP239 comA::nptII and transform BD413. The problem is that I will have to screen hundred of colonies in LB and minimal media with p-hidroxybenzoate
  2. I could extract DNA from ADP239 comA::nptII and ADP239 comP::nptII and transform SDS-6 auxotroph competent cells and select for KmR

mmm, it seems that the easiest thing to do would be the second option.