May 23Started discussing a trigger to make phage expression work. How would this mechanism be incorporated to relate to concentration gradient (QS). Some triggers include mitomycin C and phleomycin. These triggers are linked to the cellular SOS response (DNA damage). We concluded that an arabinose promoter could be used to regulate the control of a recA gene which would cleave a repressor and cause phage replication. The next step would be to find a phage that we would be able to use.
May 31Developed a team plan. The team will be taking a whole phage genome (or at least the genes that are initially ‘turned on’ when a phage begins to replicate) and cloning it into a plasmid vector. The plasmid-phage combo will be transformed into E. coli (just like you all have been doing for practice). A promoter will be placed in the phage genome (on the plasmid) that will respond or be ‘turned on’ to a signal. The signal to turn on the promoter will be the quorum sensing molecule. The E. coli will ‘sense’ the quorum sensing molecule and send a signal to the promoter OR the promoter will directly respond to the quorum sensing molecule. Once this occurs, the phage will be induced to replicate. When the phage replicates, it will hopefully lyse E. coli, and phage will be released that will lyse nearby Staphylococcus. In this scenario, the phage will need to be probably a Staphylococcus phage, since once it lyses E. coli it will need to lyse Staphylococcus, preferably a Staphylococcus phage. For proof of concept, it could be a Staphylococcus phage, but this might be harder to find.
June 4Most phages are specific for the genus (Staphylococcus vs. Escherichia) because they have a receptor to bind only to one genus (Staphylococcus) and not to other genera (like Escherichia). However, there may be other reasons that the phage will only replicate in a certain bacteria such as sigma factors, restriction enzymes present in one bacteria but not another, abortive phage proteins, and other mechanisms. However, we are not going to worry about that right now. By using the plasmid to carry the phage genome, it will be easier to manipulate the phage.
June 12A promoter that is inducible by arabinose has been found. This part is BBa_K731201. This E. coli promoter is positively regulated by arabinose. AraC is the regulator protein: in absence of arabinose the protein binds to araI1 and araO2 and the DNA forms a loop that prevents RNA polymerase from binding the promoter. When arabinose is added to the culture it binds to araC, disabling the interaction with araI1 and araO2 sites, and also allowing it to bind araI site, promoting transcription. Promoter activity is also influenced by glucose levels: in its absence cAMP levels are high and the the complex cAMP+CAP (catabolite activator protein) binds to lacI promoting transcription. In presence of glucose, cAMP levels are low and promoter activity is inhibited.
June 29The team continues to research phages that may work. A paper has been found that lists possible phages that may be used. Here is a link to the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417521/. The most promising choice for usable phage is S aureus BTN1260. This strain of S aureus's DNA encodes a phage that does not encode any virulence factors or toxins. Continued research into this possibility will be done.
July 10Discussion of how to create primers was made at the meeting. A usable phage has been found. S.aureus ATCC25923 has 5 phages in its genome, 3 are not intact so not functional, 1 is intact but has no recognizable phage genes so is probably not functional, and 1 that is probably functional - called Region 3 on the bacterial chromosome. Dr. Gentry-Weeks has this strain in and we will streak it out on an agar plate. The Region 3 phage genome is 54.1 kb. From here we will streak out the bacteria from the freezer with help from Dr. Gentry-Weeks. Note that this isolate is NOT MRSA, which is great. This is a common lab strain that is used in antibiotic susceptibility testing, so it should be good (safer) for us to use. Then we will induce the prophage to excise and replicate using mitomycin C induction – this will confirm that the phage is functional. It would be a good idea to confirm that the phage that replicates corresponds to the phage encoded in region 3. The phage DNA could be isolated and identity confirmed by PCR using primers that are specific for the region 3 prophage. We will also run tests on a different phage (from S aureus strain BTN1260) once it is received from Dr. Robinson.
July 21Developed a plan to start phage expression. Here are the parts we are going to use:
arabinose promoter BBa_I0500 ---> recA730 -- stop lambda c1 regulated promotor BBa_R0051 ---> rBBa_E0020 -- stop Constitutive Promoter BBa_J23100 --->RBS BBa_B0030 ---> cI repressor BBa_C0051 -- stop