BLAST Program Worksheet

In this example, we would look for the gapA and gapB genes that are required for the reversible conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate.

Let us look closely at the sequences obtained from plasmids from the functional genomic library of NCTC 8325 that have been found to contain a portion of a gene encoding GAPDH. The sequences of these inserts cloned into the vector is shown below with the following features:

• A portion of the sequence of the insert (the gene encoding GAPDH) cloned into the vector and set out in codons, according to the reading frame (sequence highlighted in purple)
• The TagCFP coding sequence (highlighted in teal) if shown
• The mCherry coding sequence (highlighted in red) if shown
• The remaining plasmid vector (highlighted in brown).

Functional genomic plasmid 1 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA-PURPLE) GGGGGGGGGG (ATG ………….. TAA-RED) (VECTOR SEQUENCE-BROWN)

Functional genomic plasmid 2 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA-PURPLE) GGGGGGGGGG (ATG ………….. TAA-TEAL) (VECTOR SEQUENCE-BROWN)

Functional genomic plasmid 3 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA-PURPLE) GGGGGGGGGG (ATG ………….. TAA-TEAL) (VECTOR SEQUENCE-BROWN)

Functional genomic plasmid 4 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA-PURPLE) GGGGGGGGGG (ATG ………….. TAA-RED) (VECTOR SEQUENCE-BROWN)

Functional genomic plasmid 5 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA-PURPLE) GGGGGGGGGG (ATG ………….. TAA-RED) (VECTOR SEQUENCE-BROWN)

Functional genomic plasmid 6 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA-PURPLE) GGGGGGGGGG (ATG ………….. TAA-TEAL) (VECTOR SEQUENCE-BROWN)

The partial sequences of two plasmids that contain an insert of the gene of interest cloned into a different pTH1522 functional genomics vector (= functional genomic plasmids 7 and 8) are shown below with similar features as for functional genomic plasmids 1 – 6:

Functional genomic plasmid 7 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA – PURPLE) TGAGTGAGTGAAGGAGGCTCGGG (ATG …………. TAA-TEAL) VECTOR SEQUENCE – BROWN)

Functional genomic plasmid 8 sequence

(ATG TCA ACG AAT ATT GCA ATT AAT GGT ATG GGT AGA ATT GGA AGA ATG GTA TTA CGT ATT GCA TTA CAA AAT AAA AAT TTA AAT GTA GTA GCG ATA AAT GCT AGT TAT CCA CCC GAA ACA ATT GCA CAT TTA ATC AAT TAC GAT ACG ACA CAT GGA AAA TAT AAT CTA AAA GTT GAA CCG ATT GAA AAT GGA TTG CAA – PURPLE) TGAGTGAGTGAAGGAGGCTCGGG (ATG …………. TAA-RED) VECTOR SEQUENCE – BROWN)

Q5. By using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ), identify the NCBI accession number for the BLAST hit for the gene in the Staphylococcus aureus strain NCTC8325 genome assembly Chromosome 1 that contains the insert nucleotide sequence highlighted in purple (the sequences in purple are all the same, choose one). (1.0 mark)

Q6. What is the NCBI locus tag of the gene containing the insert nucleotide sequence highlighted in purple?(1 mark)

Q7. In NCBI, what is the name of the protein product encoded by this gene? (0.5 mark)

Q8. Which of the plasmids 1 to 8 would be expected to generate a transcriptional reporter fusion at the site of the plasmid insertion that could be used to visualise fluorescence? Explain your reasoning.(5 marks)

Q9. To correlate the in silico predictions to the biological function, a plasmid could be introduced into the target NCTC 8325 strain translationally fused to either TagCFP or mCherry to visualise fluorescence.

• Which construct would you choose to visualise TagCFP inside cells and why?
• Which construct would you choose to visualise mCherry inside cells and why?