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 18th
of January. Please do not forget them
during the Christmas time.
1st) 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).
2nd) 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
