Briefly describe how you could test that the E. coli CSH45 culture is lysogenic
for λ virus CIts857Sam.
- If it contains the mutant gene and is heated to 42 C and chloroform is added, the solution will be clear with the dead bacterial cells in the precipitate
- This shows it is in the lytic cycle
- At 34 C the solution will be cloudy which means the bacterial cells are intact and so is in the lysogenic cycle.
Heat induction during the log phase of growth is necessary for efficient
production of the virus. Why is this?
- The mutation has made the CI repressor gene temperature sensitive
- CI repressor gene is normal at 34 C but at 42 C the CI mutant protein denatures
- This results in the λ phage going into the lytic cycle resulting in viral production.
If the λ virus particle can be disrupted by the addition of a Mg2+ chelator such
as EDTA, what does this tell you about the protein/protein interactions that
hold the viral capsid together?
- The viral capsid is made up of proteins held together by Mg ions.
- The electrostatic interactions is what holds the viral capsid together
- Adding a chelator e.g. EDTA binds to the Mg ions, removing it
- Results in the capsid falling apart due to the proteins not being bonded together by the Mg ions
- There is no protein-protein interactions
Given that production of a truncated S protein from the Sam7 gene of the virus
is sufficient to cause damage to the bacterial cell wall in cells in which the
virus has been produced, how can you predict that the virus has been produced
following induction of the CSH45 lysogen?
- The S protein weakens the bacterial cell wall
- Adding chloroform degrades the cell wall releasing the phages
- The solution will be more clear and more viscous as it contains high concentrations of DNA and RNA
For this experiment, what would be an appropriate control?
DH5α
- This E.coli strain does not have the mutation so can act as the control
- It doesn’t have the virus either
- Use an uninduced strain
What role does the chloroform and ethanol play in the DNA extraction from
viral particles?
Chloroform
-kills are lyses the E.coli host, releasing the phage particles into the supernatant
Ethanol
-the purified phage DNA is precipitated by ethanol
What is the electrical charge of DNA and explain how is this used in
electrophoresis? How does electrophoresis separate out DNA of different
sizes?
- DNA is negatively charged
- Electrophoresis separates DNA by charge
- DNA is put into an electrical field and run from negative to positive
- Small fragments run faster than big fragments
You want to amplify a λ viral gene in order to distinguish a lysogenic bacterial
strain from a non lysogenic strain. How could you decide which λ virus gene
would be suitable for this purpose?
CI repressor gene
-When the repressor proteins binds, the virus remains lysogenic and viral DNA replicates along side the bacterial chromosome in which it remains integrated
Why does the molar concentration of the two primers in a PCR reaction have
to be relatively high? What range of primer concentrations is typically used
for PCR?
- Need enough primers for all the template
- Each cycle will have many copies so need many copies of the primer as well
0. 1-0.5 Mm
What parameters need to be taken into account for good primer design?
- Similar melting temp
- Primer length
- Specificity
- Avoids primer dimer formation.
Why is a negative control included in the PCR?
- There should be no result, only primer dimers
- If see a band, means you have contamination and so results are not valid.
Why is PCR amplicon purification useful if the PCR amplicon is to be
sequenced?
- After PCR you still have primers (forward and reverse) and primer dimers which you don’t want
- Only need need one primer otherwise will get the wrong product.
A PCR amplification product (PCR amplicon) can be purified by a number of
methods: precipitation, electrophoresis or ExoSAP. What are we trying to
remove by doing this?
- Exosap: removal of excess dNTPs and primers and primer dimers prior to sequencing
- Precipitation: salts
- Electrophoresis: can see everything, can cut band out and repurify it e.g. melt agarose.
Why is it an advantage, in PCR reactions, for the two primers to have similar melting temperatures (Tm’s)?
- Can have only one temperature for the primers to bind to the template DNA
- Otherwise only one primer will bind in the annealing step if primers had different melting temperatures.
Briefly describe the temperatures used for the annealing, extension and
denaturation steps in each PCR cycle. Discuss why these temperatures are
chosen
95 C, 15min- Enzyme activation
95 C, 20 sec- Denaturation of DNA, to make single stranded
55 C, 15 sec- Annealing of primers
72 C, 40 sec- Extension of the primers by Taq DNA polymerase
72 C, 3 min- final amplification
What were the results from the gel electrophoresis of the PCR and do they
match with what was expected?
- Expect to see a PCR product around 700 bp
- If you have it, means you have the gene
Automated sequencing uses fluorescently labeled dideoxynucleotides
(ddNTP’s) in the sequencing reaction. How are these different to the dNTP’s
used in the PCR reaction and what role do these molecules play in the DNA
sequencing process?
ddNTPs
- Contain a dye of different colours and so each dye labelled DNA molecule can be identified because of the dye terminator at its 3’ end
- 2 oxygens have been replaced by Hs so it cant be used for elongation purposes. Act as chain terminators
dNTPs
-Used by the DNA polymerase to add nucleotides to the elongating DNA strand during replication
Briefly describe how dye-terminator sequencing works.
- Uses dNTPs and also ddNTPs
- Uses primers, DNA polymerase, dNTPs etc. for sequencing reaction mix
- During sequencing, polymerase adds complementary nucleotides to the growing chain until a ddNTP is incorporated randomly
- This terminates DNA strand extension resulting in DNA fragments of varying length
- Put into gel to analyse resulting bands
Would dye-terminator sequencing be suitable for large scale projects, such as
the human genome? Why?
No, takes too long and expensive
- Now have reversible ddNTPs and have chips which all the sequencing can be done in
- Can have thousands of sequences done at the same time, making it quicker
- At the same time of the human genome project, this was the only type of sequencing available, predicted it would take 40 yrs
Why are some of the peaks on the DNA sequencing electropherogram
different in height?
- 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.
- The height is dependant on how many copies at that particular length was made when the ddNTP was incorporated randomly
Why is it important to manually check your computer generated
electropherogram by eye?
- To check there is no gaps in the sequence
- Make sure the matches are right
- Make a consensus strand that is error free.
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
- Linear genome incorporated into an icosahedral head
- Tail fibres allow the virus to bind to the protein in the outer surface of the host
- The linear phage genome is injected past the cell outer membrane
- Then through a glucose transport protein in the inner 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.
