Methods:
The work for the semester was to be presented to the class in four modules –
Genetics, Protein Biochemistry, Recombinant DNA Methods and Development. Thus
far, the Genetics module was first completed. The process of finishing the work in that
module certainly was certainly interesting. The class first began with us, the students,
familiarizing ourselves with the characteristics of bacteria in terms of how they grow,
what they need to grow and so on. The first laboratory method that was taught was that
of streaking for singles. To execute this procedure, a toothpick was dipped into a colony
and used to inoculate the colony onto another plate. Other toothpicks were then used to
spread the cells out. With this method, I was able to separate numerous bacterial cells to
give rise to individual colonies, each arising from a single cell. Using such a method, any
researcher (or student) can select a colony of presumably one phenotype and use it for
other experiments, with some degree of confidence that it was indeed that specific
phenotype.
Following my mastery of streaking, my colleagues and I were given the
opportunity to perfect the art of serial dilutions. This method was perhaps one of the
most uninteresting, yet useful techniques in the lab. A culture solution was diluted to
give a certain concentration. This diluted stock was further diluted, again and again, until
the desired concentration(s) was/are obtained. By using this technique, I was able to
count the bacteria and estimate how much live bacteria I had started out with.
Another method of counting the bacteria was the use of the spectrophotometer.
The device measured how much light was scattered/absorbed by bacteria in liquid
medium. The use of this machine to determine the cell count had a disadvantage,
however, in that the count includes both dead and live cells. The serial dilutions, though
tedious, allowed for counting of only viable cells.
Apart from learning about how to count bacteria and separate them, I also learned
about patching. This procedure was very much like streaking but was not used to give
rise to single colonies. Instead, it was used to determine the phenotype and, subsequently
the genotype, of a particular bacterial strain after streaking for purification. It involved
the drawing of “Z”s on the plates within the squares of a grid attached to the bottom of
the plate. It was a tedious but very useful process, like counting and performing serial
dilutions, in that it allowed for the characterization of the strain.
As the semester proceeded, the art of selection was introduced. Here, the contents
of plates were manipulated so as to grow bacteria of only one phenotype. For example, I
was able to select for bacteria with resistance to the antibiotic kanamycin by growing the
bacteria on plates containing kanamycin. The technique was invaluable when trying to
deduce the phenotype and genotype of a particular strain, especially one that had its
genome altered in some way. Combined with the screening, selection was irreplaceable
in a lab where bacterial genetics was the main focus. In screening, bacteria of both the
desired and undesired phenotypes of a particular trait are grown. By observing the
phenotype of all the bacteria, the desired trait can be pinpointed. An example here would
have been the observation of red and white colonies on a MacConkey arabinose plate,
where the red signified the phenotype Ara+ (able to metabolize the sugar arabinose) while
the white signified Ara- (unable to metabolize arabinose).
The next two procedures I will describe were the methods that I used to directly
manipulate the E. coli genome: transposition/transposon mutagenesis and transduction.
Bacteriophages or phage, which are viruses that infect bacteria, are needed in these
procedures. In the first, the E. coli bacteria was infected with the phage and the insertion
of the transposons, which are DNA sequences that can essentially “hop around” in the
cell, into the bacterial genome, was brought about by manipulation of the production of a
protein, transposase, which was necessary for the process to occur. Transposons are
capable of moving from gene to gene within a certain genome or from genome to
genome.
By purification of the mutated bacteria, strains were collected where the
transposon inserts into the Ara gene, and hence converts an Ara+ LacZ- strain into an AraLacZ+ strain. (A LacZ+ strain was able to metabolize lactose while a LacZ- strain
cannot.)
Following the completion of the transposon mutagenesis process, the mutated
gene with the transposons inserted into a different bacterial strain was moved recombined
into the genome of other cells by transduction. Phage were used to break up the bacterial
genome and lyse the cells, taking with them pieces of the bacterial genome that contained
the sequence that had been manipulated. These phage were then used to infect other
bacterial strains and recombination between the genes of the recipient cell and the genes
from the lysed or donor cells was allowed. Using the data obtained from the effecting of
this technique, I was able to deduce the insertion site of the transposons.
In order to make the transposition and transduction processes as successful as
possible, a certain phage to cell ratio had to be observed in each process. To determine
this ratio, I was required to plate phage. Plating phage and bacteria are very different.
When plating phage, I had to allow them into infect the bacteria, mix the infected bacteria
in the agar and then pour the agar onto a plate. When plating bacteria, I actually had to
spread the bacteria out with a glass rod.
Whether I was plating, diluting or handling bacteria in any way, I had to observe
sterile technique always to minimize contamination. The protocol included the flaming
of the opening of every glass vessel that I removed chemicals from or put chemicals into.
I also had to light the glass rod aflame to ensure that it was sterile before spreading.
Pipette tips had to be changed constantly as well. However, the protocol had to be
observed to maintain the integrity of the results.
In the seven days that I spent working with E. coli in the Genetics module, I
learned a lot. However, I was quite content at that time to have been finally moving on to
other material, namely Protein Biochemistry. From that moment, I would have had to
spend much of my time purifying and studying the β-galactosidase, the enzyme encoded
by the LacZ gene in the E. coli genome.