In the time since our last blog post we have tried several attempts to perform a successful conjugation with multiple strains of Pseudoalteromonas and Planococcus associated with the accessory nidamental gland of the squid. We follow the conjugation protocol for the conjugation. Conjugation is how bacteria exchange genetic information. In this case our E. coli conjugates contain a transposon, which basically randomly inserts into DNA and we are hoping for this transposon to insert in the codon that codes for biofilms for our bacterial samples. Here are the basic steps from this protocol: first overnight cultures of or samples are grown. The next day we combine our intended recipient bacteria with the E. coli conjugant that has the transposon and pellet them using a centrifuge. We then resuspend the pellet and then spot the mixture of the two on a plate. We then let the bacteria grow over night at room temperature where the two should conjugate. The next day we scrape the spot off the plate, resuspend in Luria broth and pipette 10-50 microliters onto fresh plates and spread it evenly using sterile glass beads. These plates are then grown at room temperature overnight.
There is a selective antibiotic marker for erythromycin that is combine into the recipient bacterium during the transposition. This selective agent allows for us to grow up our transformed bacterial colonies. What we are hoping to get for results include interruption of the genes for biofilm and pigment formation. We will be screening for colonies with no biofilms and impaired pigment formation. However we have had difficulties with the bacterial conjugation and we have not had high numbers of bacterial transformants. Which means for some reason our bacteria isn’t conjugating with E. coli and we are trouble shooting this process.
We have recently ordered fresh antibiotic to add to the plates which should limit the growth of the donor E. coli but allow for our transformed bacteria to grow. One other factor that can be optimized to increase transformation is increasing the moisture content of the media. If this next round is not successful we may need to reorder our conjugate strains.
In the past month Dr. Wier and I have continued our research. We have sorted through our potential bacterial candidates and selected a Pseudoaltermonas strain as our focus. Pseudoaltermonas is the genus of a marine bacteria that are frequently associated with marine invertebrates. Many Pseudoalteromonas species produce pigments as well as antimicrobial compounds. We are also using a sample we are calling, “RO7.” Under a microscope I was able to observe the morphologies of the two samples as well as do gram staining. The Pseudoaltermonas sample showed to have rod morphology while our RO7 showed to be coccoid or sphere like morphology.
We recently conducted another polymerase chain reaction (PCR), which as stated in our previous post replicates and amplifies the DNA of our samples. However this time did a PCR and ran a gel electrophoresis. Gel electrophoresis is used in order to separate DNA fragments according to size. Our samples are loaded into small wells of an agarose gel. Then the gel in placed in a buffer in a electrophoresis chamber which allows current to move through the gel. As current moves through the gel, because DNA is negatively charged, DNA fragments move down the gel towards the positive end of the chamber. We will send out our amplified DNA to be sequenced in order to confirm the species of the bacteria that we are using. Our PCR was successful so we are now ready to move ahead. Next steps include making a specific media to grow our samples on in order to move forward with our protocol.
Our next step involves doing a transposon mutagenesis. We are going to take the Pseudoalteromoas strain and introduce random breaks in the genes throughout the genome in order to find genes involved in the pigments involved with antimicrobial compounds. The next stem will involve selecting many mutants and archiving them as well as screening them for different phenotypes.
For our research project we are studying marine biofilm formation. We are going to use forward genetics with transposon mutagenesis to attempt to disrupt the genes that code for biofilm formation in an attempt to better understand biofilm expression in the marine environment.
During the past month Dr. Wier and I have been trying to isolate marine bacteria that produce an easily recognizable growth pattern. The criteria include strong biofilm production and rapid colony growth. Numerous samples were taken from the local Orange Striped Anemone as well as Hawaiian water stock cultures stored in Dr. Wier’s lab. Each sample taken was inoculated in a nutrient media tube and placed in a room temperature incubator for 24-48 hours. If after this time a ring like film developed on the top of the sample in the test tube the sample was then streaked on petri plate to ensure only one bacterium was present and could be isolated.
After determining roughly 22 good bacterial candidates from our samples we then performed a polymerase chain reaction (PCR), which ultimately amplifies and copies a small piece of DNA that is used to identify our bacterial samples. The next step was to send out our DNA samples to be sequenced. When the sequences were returned to us we then used the Basic Local Alignment Search Tool (BLAST) to determine the identity of each of out isolated biofilm producing bacteria. Next steps include choosing a few appropriate candidates for the transposon mutagenesis. Now that we have a great phenotype we can break the genes in the bacterium and figure out which ones were involved in creating the biofilm.