Presentation Flashcards
Lambda phage
-Temperate phage: uses both lytic and lysogenic cycle
-Bacteriophage are viruses that can infect and destroy bacteria
-Double stranded DNA virus of E.coli
protein in the outer surface of the host
-The linear phage genome is injected past the cell outer membrane
-In the cytoplasm the DNA circularises
Lytic cycle
- The ƛ phage injects its DNA into the host
- Phage proteins are expressed and take over protein synthesis and DNA replication machinery of infected cell
- Phage DNA replication occurs
- Phages are replicated many times, leading to the production of new phages within the cell.
- Infected cell burst (lyses) releasing the new viral particles which go on to infecting other cells.
Proteins maintaining lytic cycle
- Cro and S proteins.
- The lytic cycle occurs when conditions are favourable for growth thus cellular proteases are active; the proteases degrade cII protein resulting in a decrease of the repressor protein.
- This leads to greater expression of the cro protein, responsible for the lytic cycle. Lysis of the cell is due to the expression of the S protein which encodes a lysozyme enzyme that weakens and degrades the host’s cell wall.
Lysogenic cycle
- Under unfavourable conditions, the phage DNA can integrate itself into the host’s genome and enter the lysogenic cycle.
- In this state, the phage is called a prophage which stays inside the host cell causing no harm until conditions are favourable.
- Does not result in immediate lysing of the host cell
- Stays dormant until environmental conditions increase so then divides, lyses and infect more cells.
- Lysosgenic cycle allows the host cell to survive and reproduce allowing the virus to be reproduced in all of the offspring.
Proteins maintaining lysogenic cycle
- The prophage expresses proteins that act to repress expression of proteins important for the lytic cycle; the most important is the cI protein.
- It encodes a repressor protein which binds to OL and OR which inhibits RNA polymerase from initiating transcription at PL and PR thus repressing transcription of the viral early genes (N, cIII, cro, cII, (O,P,Q replication protein) to inhibit the lytic cycle.
- The repressor protein also acts as an activator of PRM to activate the production of cI.
- The viral DNA is integrated into the host chromosome and replicates with it.
- No new viral phages are produced and the virus is only transmitted vertically.
Intro to method
- In this experiment we used the E.coli strain CSH45 which contained a ƛ virus mutant,
- manipulate the virus to enter the lytic cycle and control its release from the host.
- E.coli strain DH5𝛂 was also used as a control.
Method 1
Aim: To Isolate and purify ƛ virus DNA from virus particles and genomic DNA from E.coli strains CSH45 and DH5𝛂
-Lysed induced CSH45 using chloroform and precipitated the ƛ viral particles in alcohol buffer.
-Chloroform kills and lyses the host, releasing the phage particles into the supernatant.
-Viral DNA was extracted from the particles using Viral Disruption Buffer, isopropanol and ethanol.
-Proteins released with DNA are precipitated by salt and chloroform, leaving the purified phage DNA to be precipitated by ethanol
Genomic DNA was isolated using a common isolation method.
The viral DNA was loaded onto a gel. This is shown in figure 2. This was to make sure that the DNA isolated was indeed the viral DNA.
Method 2
Aim: PCR amplification of the CIII gene from the isolated ƛ virus and from genomic DNA from E.coli.
- 4 PCR reactions were set up containing:
1. Viral DNA (V)
2. Genomic DNA from CSH45 (C)
3. Genomic DNA from DH5𝛂
4. No DNA (negative control) - ExoSAP mix was added to tubes C and V to remove excess dNTPs and primers prior to sequencing.
- This was then added to the cycle sequencing reaction mix that contains 2 primers that flank the CIII gene and ddNTPs.
- The C and V template DNA was added into 2 PCR tubes each, one was the forward primer and the second was the reverse primer.
- Dideoxy terminator nucleotides stop synthesis when incorporated into a molecule.
- Each ddNTP contains a dye of a different colour
- Each dye labeled DNA molecule can be identified because of the dye labeled terminator at its 3’ end.
- The molecules are then separated by capillary electrophoresis allowing the sequence of the DNA to be determined.
Method 3
Aim: Analyze of the DNA sequence from the electropherograms and the nucleotide and protein sequences using bioinformatics.
The C and V amplified DNA was sent for sequencing.
- Alignment of sequences obtained from DNA sequencing results were used to create a consensus sequence.
- BLAST search to find regions of local similarity between sequences. To see whether the sequence matches the one in the database
- We did an Open Reading Frame-finder Analysis to find all the open reading frames of the gene it encodes
- Designed primers of amplification of viral products.
Agarose Electrophoresis gel
- The PCR product was run on an agarose electrophoresis gel. The DNA is put into an electrical field, which separates DNA based on size since DNA is negatively charged.
- From the gel electrophoresis shown in figure 3 of the PCR product we can see a product around 700 bp. This results means we have the CIII gene.
Electropherogram
- The peaks in the middle are well defined and sharp with little background noise. This means that the sequence will be considered accurate from a visual perspective.
- The start and end of the electropherogram is where the probability of the inclusion of a ddNTP is low. This means there are fewer short and very long PCR products.
- Due to the lower PCR product amount, the fluorescence is smaller and the peaks are less defined.
- A consensus strand was made where these were cut out in order to make an error free sequence.
CIts857 mutation
- Responsible for making the cI protein more sensitive to temperature.
- The cI protein is responsible to keeping the prophage in the lysogenic state, and so making it temperature sensitive allows us to easily denature it at 42 degrees.
- The induced strain used had gone through heat treatment therefore the cI protein had been denatured and the virus went into the lytic cycle.
- This means that it will start replicating and be ready to be released into the environment.
Sam 7 mutation
- Affects the S gene which normally encodes a lysozyme enzyme that degrades the cell wall allowing release of the phages.
- The mutation causes the enzyme to only weaken the cell wall by making partially functional proteins and not allow it to lyse until treated with chloroform.
- Gives us control of the cell by deciding when to lyse it so we can get more phages replicated.
CIII
Responsible for stabilising the cII proteins which activates transcription from PRE to produce repressor proteins, helping to maintain the lysogenic state.
