BCMB 301B Lab 4 Transposition Insertion Mutagenesis (mini-kan and lambda phage)

What are some reasons for insertion specificity of transposable elements?

target sites or hotspots in genome may conform closely to a consensus sequence used by transposase

What are the 3 types of transposable elements in bacteria?

simple insertion sequences (IS)

Composite transposons (Tn10 or mini-kan)

Large complex transposable bacteriophages

What is an IS element?

“Insertion sequence element” that contains transposase genes flanked by two Inverted repeats (IR)

What are inverted repeats (IR)?

they are binding sites for transposase

What are composite transposons composed of?

They are flanked by IS elements with other genes in the central region

What are the two mechanisms of transposition?

replicative - copy and paste

Conservative - cut and paste

What enzyme mediates the recombination in transposition?

the transposase enzyme (independent of the cell’s RecA system)

What in mini-kan derived from?

the naturally occurring Tn10 transposon

How has mini-kan been altered from Tn10 and why?

• removed a copy of the transposase and moved the other into the viral DNA and under control of the Ptac promoter (allow for the control of expression of transposase using IPTG/lacI and prevent the insertion to transposase into the genome which would otherwise be available to preform reversion = increase in Transposition Frequency)

• Removed the IR internal sequences recognized by transposase (prevent the possibility of inverse transposition)

• tn10 tetracycline resistance genes have been replaced by tn903’s kanamycin (may be due to an antibiotic stability issue)

Where does tn10 transposase cut?

• it creates precise double strand breaks within the IR sequences (external repeats are bound preferentially) in the IS elements

• It creates single stranded nicks in the 9bp target sequence DNA —> insert and DNA Poly I fills in the gaps

What is the mariner class system?

it is a class of transposons found in prokaryotes and eukaryotes (most widely used today)

How does TnSeq work ?

• sequence a pool of random transposon mutants

• apply the conditions of interest (to ID genes required for survival)

• sequence the remaining mutants to compare with what was initially there with the purpose of identifying genes important for survival

What are some other ways transposons can be delivered?

plasmid transformation

virus through transduction

electroporation

What is the mechanism of attachment of lambda bacteriophage to E. coli?

• it attaches to the maltose receptor on the bacterial membrane using Mg+2 to stabilise the interaction

How do we improve the absorption efficiency of the phage into E. coli?

supplement the growth medium with maltose to upregulate the number of attachment sites and supplement with Mg +2 to stabilise the interaction

What determines the Lambda lytic cycle will occur?

if the replication of the LATE stage structural genes occurs before the production of the lambda CI repressor

What determines the Lambda lysogengic cycle will occur?

if the CI repressor is produced before the late stage structural genes

What does the CI repressor do?

it promotes the lysogenic cycle by binding an operator site on the phage genome which results in the repression of the structural genes and the promotion of the cI phage genes

What are lysogens?

cells that have the viral DNA inserted in their genome (this includes the cell’s progeny)

What is the Ptac promoter and what does it do in this lab?

• it is STRONG hybrid promoter that is a fusion of the trp operon promotor and the lac operon promoter/operator that can be induced by the addition of IPTG to remove LacI

• in this lab it controls the expression of the transposase (not the lac operon expression for the differentiation of Lac- mutants)

What type of transposon is mini-kan?

conservative (cut and paste)

What does the nin5 deletion do?

it removes around 2.5 kb of lambda 1105 phage DNA to allow the mini-kan with the remaining viral genome to be packaged successfully in the viral head

What are the 2 conditional mutations in lambda 1105? Why is it important to have these in this experiment?

• cI repressor temperature sensitive mutation (is not stable at temperatures above 39.5 C and therefore won’t enter the lysogenic cycle)

• nonsense amber mutation in the P gene UAG stop codon (amber codon) introduced (can’t complete the lytic cycle without this late stage structural gene)

• this allows for the phage to act as a suicide vector to deliver the mini-kan construct to E. coli W3110 without lysis

What is an amber suppressor mutation?

• it is a mutation found in a specific tRNA coding region in the genome

• the tRNA now binds UAG stop codon to deliver it’s amino acid instead which results in a read through

How do E.coli survive amber suppressor mutations?

the UAG stop codon is the least utilised stop codon in the genome therefore a read through has the least effect out of all the other stop codons used

What is the amber suppressor mutation(s) in E. coli BNN45 ?

• a change from recognition of UAC encoding TYROSINE to a recognition or UAG (a stop codon) to deliver TYROSINE

• a change form CAG GLUTAMINE insertion to using UAG to deliver GLUTAMINE

Why is it important for BNN45 to have the amber suppressor mutations?

The lambda 1105 phage has a nonsense amber mutation in its P gene resulting in an early stop codon (can’t complete the lytic cycle) BUT BNN45 can read through this mutation and rescue it. This allows for the phage to be titred (MOI calculation) before it is used as a suicide vector for the mini-kan construct into W3110 for transposition mutagenesis.

What is a plaque assay?

it is an indirect assay used to quantify the number of viral particles

• incubating the diluted phage with a susceptible bacterial host

• mix with molten agar and pour on nutrient agar plate

• incubate

• a plaque is formed when a viral particle infects and undergoes the lytic cycle resulting in cell lysis

What does plaque morphology tell us about the bacteriophage?

• clear plaques = can under go lytic cycle that results in cell lysis

• turbid plaques = can undergo lytic and lysogenic cycles (a few number of living lysogen remain)

• size of plaque is dependent on phage, bacterial host and the culture conditions

Why do we calculate the titre of the phage (pfu/mL)?

it is used in the MOI calculation

What is MOI? Where is the target value for transposition mutagenesis and why do we need it?

• multiplicity of infection or the # of pfu/cfu

• we want this value to be between 0.05-0.5 to prevent more than one phage from attaching to a bacterial cell (attachment of too many phage particles can destabilise the membrane causing premature cell lysis)

What strain must be sensitive to Kanamycin in this lab? Why?

E. coli W3110 because it allows for the selection for cells that have acquired the transposon KanR genes and therefore the mini-kan transposon

What is a polar mutation?

it is a mutation (insertion in our lab) in an operon gene that can disrupt the transcription of the genes downstream

e.g. lacZ is ahead of LacY in the lac operon

What does LacY encode? What does it do?

Lac permease allows for the entry of lactose into the cell

What does LacZ encode for?

B-galactosidase

What does Lac-Tz medium have added to it and why?

• lactose and tetrazolium

• allow for the growth of Lac+ and Lac-

• tetrazolium is reduced to form a red insoluble formazan when in the presence of neutral pH Lac- colonies and this can be used to differentiate between the acidic Lac+ (clear)

What is tetrazolium reduced to in the presence of Lac- mutants?

formazan (red and insoluble)

What is the importance of the Lac-Tz + Kan media?

It is both selective and differential

• selects for mini-kan using the kanamycin

• differentiates Lac- (@neutal pH=insoluble red formazan) and Lac+ (clear) phenotype

Is Lac permease required for IPTG or X-gal entry into the cytosol?

No. (This allows us to differentiate between lacYmutants vs LacZ)

What is the plate and additives for the characterisation of Lac- mutants?

Luria Bertani (LB) agar with IPTG (remove lacI from lac operon operator) and X-gal (cleaved by B-gal if LacZ is functional)