Team:Technion/Project/Phage

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Objective

The main objective of this project is to create phage lambda that goes through its lytic cycle only under specific conditions that are met in the bacterial host. The idea is to replace one phage protein location in the genome, under a new regulatory promoter. This will allow the phage lytic cycle only in inducible conditions, controlled by the engineered plasmids that express RNA-polymerases.
This project included planning the genetic manipulation of the phage genome. This includes:

• Phage deviation into fragments that will ease the genetic manipulation, and re-factoring of the phages genome, after cutting it into fragments.
• Planning of the Q gene deletion and re-insertion under the desirable regulation, the RNA-polymerase promoter.
• The design of the antibiotic resistance gene insertion into the phage genome, in order to create additional selection to bacteria that contain the phage lysogenic genome.

The chosen phage lambda strain

The phage that was chosen as our working tool was phage lambda heat – inducible cI857s7. The phage genome contains four mutations:

• Addition of HindIII restriction site at 37,589 (C -> T) [ind1].
• Mutated S gene at 45,352 (G -> A), which leads to accumulation of infectious bacteriophage in the E. coli cells, the phage concentration increase when released from the cell [Sam7].
• Temperature sensitive mutation that converts the CI gene into a thermo sensitive protein. This allows inducing the lysogenic phage cycle in 37˚C, and lysis induction in 42˚C, this mutation is created at 37,742 (C -> T) [cI857].
• Additional point mutation at 43,082 (G -> A).

The phage genome sequence was taken from [http://www.ncbi.nlm.nih.gov/nuccore/NC_001416.1 NCBI]
The physical DNA was obtained from [http://www.neb.com/nebecomm/products/productn3011.asp NEB]
We chose to work with this phage mainly because of the temperature sensitivity, and the ability to induce lysis in controlled conditions. Moreover, the phage concentration will be higher when the bacterial cell undergoes lysis, due to the Sum 7 mutation.

The division into fragments

When thinking on how to manipulate the phage genome and insert one or more genes under desire regulation (like RNA-pol promoters in our study), the first problem is how to manipulate a 48kb genome.
Our solution for this problem is to divide the phage genome into eight fragments.
Figure 1 shows the whole phage genome as divided into fragments.

Figure 1: A – phage lambda genome with notations to the regulatory sequences (promoters and operators), regulatory proteins, functional proteins and structural elements. The colored frames represent the divided fragments, with no significance to the different colors. Each letter or word above the sequence represents the different genes. The colored genes consists the phage envelope, and their location in illustrated in B. B – phage lambda structure illustration. The proteins’ colors match the colored sequences in A.
The original figure was taken from: S.V. Rajagopala1, S. Casjens. and P. Uetz, 2011, "The protein interaction map of bacteriophage lambda", BMC Microbiology, 1, pp. 213-228.