Lambda lab

Question 1

Briefly describe how you could test that the E. coli CSH45 culture is lysogenic
for λ virus CIts857Sam.

Answer 1

• 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.

Question 2

Heat induction during the log phase of growth is necessary for efficient
production of the virus. Why is this?

Answer 2

• 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.

Question 3

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?

Answer 3

• 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

Question 4

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?

Answer 4

• 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

Question 5

For this experiment, what would be an appropriate control?

Answer 5

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

Question 6

What role does the chloroform and ethanol play in the DNA extraction from
viral particles?

Answer 6

Chloroform
-kills are lyses the E.coli host, releasing the phage particles into the supernatant

Ethanol
-the purified phage DNA is precipitated by ethanol

Question 7

What is the electrical charge of DNA and explain how is this used in
electrophoresis? How does electrophoresis separate out DNA of different
sizes?

Answer 7

• 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

Question 8

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?

Answer 8

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

Question 9

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?

Answer 9

• 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

Question 10

What parameters need to be taken into account for good primer design?

Answer 10

• Similar melting temp
• Primer length
• Specificity
• Avoids primer dimer formation.

Question 11

Why is a negative control included in the PCR?

Answer 11

• There should be no result, only primer dimers

- If see a band, means you have contamination and so results are not valid.

Question 12

Why is PCR amplicon purification useful if the PCR amplicon is to be
sequenced?

Answer 12

• 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.

Question 13

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?

Answer 13

• 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.

Question 14

Why is it an advantage, in PCR reactions, for the two primers to have similar
melting temperatures (Tm’s)?

Answer 14

• 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.

Question 15

Briefly describe the temperatures used for the annealing, extension and
denaturation steps in each PCR cycle. Discuss why these temperatures are
chosen

Answer 15

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

Question 16

What were the results from the gel electrophoresis of the PCR and do they
match with what was expected?

Answer 16

• Expect to see a PCR product around 700 bp

- If you have it, means you have the gene

Question 17

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?

Answer 17

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

Question 18

Briefly describe how dye-terminator sequencing works.

Answer 18

• 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

Question 19

Would dye-terminator sequencing be suitable for large scale projects, such as
the human genome? Why?

Answer 19

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

Question 20

Why are some of the peaks on the DNA sequencing electropherogram
different in height?

Answer 20

• 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

Question 21

Why is it important to manually check your computer generated
electropherogram by eye?

Answer 21

• To check there is no gaps in the sequence
• Make sure the matches are right
• Make a consensus strand that is error free.

Question 22

Lambda phage

Answer 22

• 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

Question 23

Lytic cycle

Answer 23

• 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.

Question 24

Proteins maintaining lytic cycle

Answer 24

• 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.

Question 25

Lysogenic cycle

Answer 25

• 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.

Question 26

Proteins maintaining lysogenic cycle

Answer 26

• 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.

Question 27

Intro to method

Answer 27

• In this experiment we used the E.coli strain CSH45 which contained a ƛ virus mutant, CIts857Sam7, to 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.

Question 28

Method 1

Answer 28

Aim: To Isolate and purify ƛ virus DNA from virus particles and genomic DNA from E.coli strains CSH45 and DH5𝛂

• Chloroform kills and lyses the host, releasing the phage particles into the supernatant.
• The phage are precipitated with an alcohol buffer
• The isolated phage is lysed with GITC
• Proteins released with DNA are precipitated by salt and chloroform, leaving the purified phage DNA to be precipitated by ethanol

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.

Question 29

Method 2

Answer 29

Aim: PCR amplification of the CIII gene from the isolated ƛ virus and from genomic DNA from E.coli.

PCR and sequencing the CIII gene.

• PCR amplicons were run on an agarose gel to see if the reaction was successful
• 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.
• The primers anneal to complementary sequences in the template and primes multiple rounds of DNA synthesis to produce billions of single stranded copies of the selected DNA.
• The reaction mix contains dideoxy terminator nucleotides that will stop synthesis when incorporated into a molecule.
• Each ddNTP contains a dye of a different colour and therefore 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 with one base pair resolution allowing the sequence of the DNA to be determined.

Question 30

Method 3

Answer 30

Aim: Analysis of the DNA sequence electropherograms and nucleotide and protein sequences using bioinformatics.

The C and V amplified DNA was sent for sequencing.

• Alignment of sequences obtained from DNA sequencing results to create a consensus sequence.
• BLAST search to find regions of local similarity between sequences.
• ORF-finder Analysis.
• Designed primers of amplification of viral products.

Question 31

Agarose Electrophoresis gel

Answer 31

• 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.

Question 32

Electropherogram

Answer 32

• 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.

Question 33

CIts857 mutation

Answer 33

• 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.

Question 34

Sam 7 mutation

Answer 34

• 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.

Question 35

CIII

Answer 35

Responsible for stabilising the cII proteins which activates transcription from PRE to produce repressor proteins, helping to maintain the lysogenic state.