Determining our basal mutation frequency

Hi All:

After “playing” to be microbiologist of previous sessions, yesterday we began to deep in our project dealing with “the determination of mutation frequency of a bacteria under ultraviolet (uv) pressure”.

First of all, remind all the things we performed yesterday.We have to take them in mind for our next session on 18^th of January. Please do not forget them during the Christmas time.

1^st) From previous bacterial experiments we determine that the best house to obtain bacterial growth data is the Paula’s home. In order to compare future experiments and data we decided to use as growth chamber the drawer of her room. We need a picture how all these plates are incubated (see previous blog as example: http://dondemutanlosgenes.blogspot.com.es/2015_01_01_archive.html).

2^nd) Single colonies of the bacteria 3I and 3G were inoculated on Monday (two days before; I made it!!) in order to determine how many bacteria present a mutation in the SacB gene (the basal mutation frequency). However, only 3G grew. In science, sometimes occur“these things”. We decided to collect data of three independent colonies (1-3) of the bacteria 3G. To perform this, we:

 a)      First, measure the OD₆₀₀ of the culture and consider that 0.5 corresponds approximately to 10⁸bact/ml.

 

1- Cristina collecting the OD data which are:

1: 0.656

2: 0.377

3: 0.620

b)      We assume that all cultures contain similar amount of bacteria to measure the mutation frequency (but we expect that two of them the numbers must be higher, aren´t they?). In order to estimate such number we prepare plates of TY media containing Kanamycin as antibiotic and also with and without 5% of Sacarose.  Following a rule of serial dilutions:

2- Serial dilutions performed

We get three groups of plates corresponding with every single culture (1-3). And from the culture 3 we get a “duplo” of plates incubated in the Lab (our control).

c)       In 6-7 days (maybe less) we expect to have the data of single colonies to count (four data to get the mutant frequency). In order to understand what we are performing we can follow all the entries of the previous blog: http://dondemutanlosgenes.blogspot.com.es/.

Going from the first entries: Nov 2014 untill the end.

Our methodology is similar… .

But, what is different, what is new?

We performed yesterday our first uv shock test in order to “calibrate” the uv pressure for next experiments.

In resources you have a manuscript describing the recA mutant in S. meliloti bacteria (article link). We must understand that a bacteria with this gene mutated is more sensitive to uv treatment than the wild type (see figure 6A of the article).

3-Effect of uv treatment on RecA mutant bacterial cells

Then, we compare in our hands this test by spreading a continuous lawn of bacteria (wt and RecA mutant) on plates and defining a time course from 10’, 5’,2’,1’, 30’’,10’’ and 5’’ of uv treatment.

 4-Preparing the plate for time course of uv treatment

 1-video: how the bacterial extension was made.

 5- uv shock

Again, a “duplo” was made and one of the plates is at Paula’s home meanwhile the other is in the Lab. We have to decide which is the range of the uv shock to perform our mutagenesis experiments.

Be aware (mails, blogs and wassaps) of our first results of this fantastic project!!!

And Merry Chirstmas !!

Francisco Martínez-Abarca

Departamento de Microbiología y Sistemas Simbióticos.

Estación Experimental del Zaidín. CSIC.

Granada