1. The soil bacterium Pseudomonas fluorescens (taxid:294)is heavy metal tolerant and can survive in industrial waste anddegrade and detoxify diverse organic pollutants.

You obtain Pseudomonasfluorescens ATCC 13525 which is sensitive to ampicillin (Ap),streptomycin (Sm) and spectinomycin (Sp) but resistant tochloramphenicol (Cm) and nalidixic acid (Nx). This strain is alsolacZnegative (devoid of β-galactosidase activity).

You also obtain a donorEscherichia coli strain containing an ampicillin (Ap)resistant suicidal plasmid carrying the minitransposonmTn5-lacZSmSp  (this mTn5 has apromoterless lacZ and expresses Sm and Sp resistance).This strain is not resistant to any other antibiotics.

Consider in this case that cells candevelop resistance to each antibiotic gene through spontaneousmutation at the frequency of 1 in 10⁶ cells.

You conjugally mate 10⁹cells of ATCC 13525 with 10⁹ cells of the E.coli donor strain to construct random mTn5 mutationsand identify molecular determinants for cadmium resistance.

1. What combination of antibiotics would you use in the media toselect specifically for ATCC 13525::mTn5-lacZSmSptransconjugants and prevent the growth of spontaneous antbioticresistantmutants?                                                       

1. To prevent secondary mutations from plasmid insertion, youwould need to ensure the transconjugant cells had lost the deliveryplasmid. How would you confirm the delivery plasmid was no longerpresent in thesemutants?                        (1mark)

You then replica patch transconjugantsonto media containing the chromogenic substrate X-Gal in thepresence and absence of cadmium. The following table shows thephenotypes of selected transconjugants.

Transconjugant number	Colour of colony after growth (β-galactosidase expression)
Media without cadmium	Media with cadmium
1	White	No growth
4	Deep blue	Deep blue
9	Deep blue	Light blue
11	White	Medium blue
12	Medium blue	Dark blue
17	White	Deep blue
20	Light blue	Deep blue

c. Which mutant(s) have insertions in genes essential forcadmium resistance?

d. Which mutant(s) have insertions in genes up-regulated bycadmium?

e. Which mutant(s) have insertions in genes down-regulated bycadmium?

f. Which mutant(s) have insertions in genes that areconstitutively expressed?

g. Which mutant(s) have insertions in genes that are notexpressed in both the absence and presence of cadmium?

h. The DNA flanking the mTn5-lacZSmSp mutation wascloned from the cadmium sensitive mutant and the following sequencewas obtained for the coding strand:

                

         5’..GGCAGCACCAGCAAGG mTn5-lacZSmSp CCAGCAAGGTCAGCA..3’

Using this sequence provided,reconstruct the wild-type sequence

i. Provided with the information that the 5’ second base(underlined) in the provided sequence is the first base of a codon,provide the wild-type amino acid sequence of this portion of theprotein and identify the amino and carboxyl termini.

j. Using the established amino acid sequence and the BLASTalgorithm, what is the E value and the accession number of thehighest scoring hit that aligns in the NCBI GenBank database?

k. What can you deduce to be the function of the protein basedon the alignments obtained using the BLAST algorithm? (1 mark)