Introduction to Bacteriophage λ
Working with λ (-can someone type up this page, most likely pradeep as this is your main project goal. Brief discussion of amber mutants/suppressors, titering)
The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project:
As the recombineering, testing of riboregulators, and titering processes will take place concurrently, we needed to find a simpler way to regulate viral protein concentrations in the cells. To this end, E. coli strains have been constructed that contain a low-copy plasmid construct where one of three key developmental viral genes - coding for the cro, N, or Q proteins - is regulated by a tetracycline-dependent promoter. A constitutive promoter (J23100, the stronger promoter, or J23116, the weaker one) produces a steady stream of tetracycline repressor (tetR), which substitutes for the cis repressor in repressing protein levels. The addition of anhydrotetracycline (aTc, acting as the trans activator) inactivates the tetracycline repressor and leads to the production of the respective viral protein in the E. coli cells. This allows us to control the concentration of viral protein produced in the cells by adding varying amounts of aTc to the bacterial growth media.
Titering experiments where cro, N, and Q amber phages were allowed to infect D1210 cells containing the built construct show that heterologous N and Q can complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q even further, as it eliminated lysis completely. We were unable to express sufficient cro from a plasmid to rescue lytic behavior of the amber cro mutant phage.
Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. So far, cis construct number 3 and its accompanying trans combinations (cis3trans1 and cis3trans2) seem the most promising. Phages resulting from the recombineering process are also being screened for successful N and Q amber mutants.
The future of the project lies in confirming trans activation: that is, to prove protein concentration is several times greater in aTc-saturated trans-containing cells as compared to aTc-saturated strains with no trans plasmid. The successful complementary cis-trans pairs can then be incorporated into the N-J23100 and Q-J23116 constructs. Cis repression in the N-construct has yet to be tested, but Q’s results would imply successful repression of lytic action. Based on the presence of plaques on D1210-N and D1210-Q strains even without aTc, a small amount of trans-activation would be enough to let the amber infecting phage choose the lytic pathway.
Cis repression of the N and Q constructs can be tested by further titering. A quantitative measure of the increase in protein concentration upon trans-activation will be obtained by fluorescence measurements by flow cytometry. So far, trans activation has been shown in YFP Quanta experiments (cis3trans1 and cis3trans2 seem the most promising), although combinations remain that need to be tested. The ultimate test, though, lies in titering amber mutant phage into strains containing both cis and trans plasmids. A successful result (no plaques on cis containing strains, but plaques on cis-trans strains) would indicate a successful integration of the two independent components (N/Q protein dependent lysis switch, and the “lock and key” riboregulator) of the project, demonstrating that standardization on multiple levels: in the basic BioBrick parts making each construct, and the more abstract merging of riboregulators into viral decision-making.
Our current top priority, then, is to transform the trans1 and trans2 plasmids into the cis3-Q construct cell, titer and check for the presence of plaques. If successful, we can being the final integration steps, moving the construct entirely into the recombineered amber phage. In the final system, infecting amber λ-zap virus will selectively lyse those cells that contain a trans component in their genome, as the trans mRNA in the cell would allow the phage to inactivate cis-hairpin transcripts, and allow the cell to produce the viral proteins necessary for lysis.