1. You want to do some studies on the structure of the Yersinia pestis (bubonic

Question

1. You want to do some studies on the structure of the Yersinia pestis (bubonic plague) F1 proteins, which have been shown to be required for transmission of the Y. pestis bacteria from fleas to mice. You therefore want to use a high copy number vector to clone the coding sequence of the caf1A gene into E. coli, and make a lot of the protein, which you will then purify from E. coli. Go to GenBank, and look up the sequence of the caf1 operon, which contains the genes that code for four F1 proteins, caf1R, caf1M, caf1A, and caf1 (accession number AY450847.1). Then design 20 nucleotide long primers to amplify the caf1A gene so that the primers are within 200 basepairs of the start and stop codons at each end of the coding sequence of the gene. The PCR product should contain the start and stop codons for caf1A (the RBS is not annotated in this entry, but by including at least 50 basepairs upstream of the start codon, it should be included). You can design the primers by eye or you can use an online program at http://www.ncbi.nlm.nih.gov/tools/primer-blast/, which is easy to use (but you will need to change some of the preset parameters). Make sure the primers you design have Tms of at least 45oC and that they are within 5oC of each other. Copy the sequences of your primers and their Tms into your homework.

2. Now Google

Explanation / Answer

Range 10: 4663 to 4669 GenBank GraphicsNext Match Previous Match First Match

http://www.ncbi.nlm.nih.gov/nuccore/39754771?report=graph&rid=EG3NUBYN114[39754771]&tracks=[key:sequence_track,name:Sequence,display_name:Sequence,id:STD1,category:Sequence,annots:Sequence,ShowLabel:true][key:gene_model_track,CDSProductFeats:false][key:alignment_track,name:other%20alignments,annots:NG%20Alignments|Refseq%20Alignments|Gnomon%20Alignments|Unnamed,shown:false]&v=4663:4669&appname=ncbiblast&link_loc=fromHSP

link contains grahical representation of the forward primed PCR product which is 170bp long.

Range 6: 5163 to 5170 GenBank Graphics Next Match Previous Match First Match

http://www.ncbi.nlm.nih.gov/nuccore/39754771?report=graph&rid=EG4EFJSK114[39754771]&tracks=[key:sequence_track,name:Sequence,display_name:Sequence,id:STD1,category:Sequence,annots:Sequence,ShowLabel:true][key:gene_model_track,CDSProductFeats:false][key:alignment_track,name:other%20alignments,annots:NG%20Alignments|Refseq%20Alignments|Gnomon%20Alignments|Unnamed,shown:false]&v=5163:5170&appname=ncbiblast&link_loc=fromHSP

the above link gives graphical representation of reverse primed PCR product which is 170bp.

Primers for caf1 protein of yersinia pestis.

Forward primer: 3

Alignment statistics for match #10 Score Expect Identities Gaps Strand 14.4 bits(7) 3.2 7/7(100%) 0/7(0%) Plus/Minus

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