Curtin University Pathway Development Inactivation Worksheet

Common features of pTH1522 derivatives

An origin of transfer: oriT (not shown in the plasmid maps)

Promoterless reporters: lacZ and gusA (colourimetric reporters), TagCFP and mCherry (fluorescent reporters).

Specific features of pTH1522 derivatives

Plasmid A:

Origin of replication: pMB1 (replicates in E. coli and a few close relatives only)

Antibiotic marker: mecA methicillin resistance

Plasmid B:

Origin of replication: pMB1 (replicates in E. coli and a few close relatives only)

Antibiotic marker: aadA spectinomycin resistance

Plasmid C:

Origin of replication: pMB1 (replicates in E. coli and a few close relatives only)

Antibiotic marker: aadA spectinomycin resistance

Stop codons in three reading-frames upstream of the first reporter gene on either side of the XhoI site

Plasmid D:

Origin of replication: pAMβ1 (broad host range oriV, able to replicate in Gram-positive bacteria)

Antibiotic marker: aadA spectinomycin resistance

Plasmid E:

Origin of replication: pAMβ1 (broad host range oriV, able to replicate in Gram-positive bacteria)

Antibiotic marker: aadA spectinomycin resistance

Stop codons in three reading-frames upstream of the first reporter gene on either side of the XhoI site

Plasmid F:

Origin of replication: pAMβ1 (broad host range oriV in Gram-positive bacteria)

Antibiotic marker: mecA methicillin resistance

Stop codons in three reading-frames upstream of the first reporter gene on either side of the XhoI site

Questions relating to all the above information

Q1.Give reasons for which of the six derivatives of the pTH1522 functional genomics vector (Plasmids A to F) you would choose if you wanted to create:

i) A transcriptional fusion of the gene of interest in a functional genomics plasmid that could be inserted into the genome of S. aureus NCTC 8325 (3.0 marks)

ii) A translational fusion of the gene of interest in a functional genomics plasmid that could be inserted into the genome of S. aureus NCTC 8325 (3.0 marks)

Q2. i) What is the purpose of the antibiotic marker in your chosen plasmid(s)?(1.0 mark)

ii) What is the full name of the enzyme encoded by aadA that provides resistance to spectinomycin? (1.0 mark)

iii) Describe how this AadA enzyme inactivates the antibiotic. (1.0 mark)

iv) Describe how the mecA encoded enzyme inactivates the antibiotic. (1.0 mark)

Q3. Why would you not use the colourimetric promoterless reporters in the target strain?

Background information

While many bacteria contain a single GAPDH gene, some have multiple functional homologues. Staphylococcus aureus is a Gram-positive, coccoid bacterium that forms part of the human microbiota and is usually a commensal organism but can also become an opportunistic pathogen. S. aureus is a facultative anaerobe that can grow without the need for oxygen. It contains two GAPDH homologues, encoded by the genes gapA and gapB. The GapA and GapB proteins share approximately 40% amino acid sequence identity Previous studies have shown that gapA and gapB may be important in the pathogenesis of S. aureus.

You want to use a functional genomics approach to characterise the roles of gapA and gapB in S. aureus metabolism. To do this, you will construct a functional genomics library using DNA from S. aureus strain NCTC 8325 (IMG Genome ID = 637000279).

The library will be constructed by ligating 2 kb DNA fragments into a functional genomics vector. For this you will use a derivative of pTH1522, which can be mobilised but which cannot be replicated in the recipient cell. This vector also has divergent promoterless reporters flanking the cloning site in the vector.

By using this plasmid, we can not only create single crossover (SXO) mutants in the recipient but we can also monitor gene expression at the site of insertion. The single crossover would generate a reporter fusion at the site of the insertion.

Attributes of the bacterial donor and recipient strains that need to be considered.

We need to consider the properties and the genetic background of 1) the E. coli strain that we will use to create the functional genomics library, and 2) the target strain (S. aureus strain NCTC 8325) to make sure we choose an appropriate functional genomics vector.