Eric Olsen - CHEM215 - Problem Set 1 - 1/19/2
Q1: Visualizing E. coli gene sequences.
Look up the E. coli NNR gene in NCBI. What is its full (non-shorthand) gene name?
What are the names of its two neighboring genes (list the shorthand and full gene
names)?
>full name - NAD(P)HX epimerase/NAD(P)HX dehydratase
>adjacent genes are tsaE (N(6)-L-threonylcarbamoyladenine synthase, TsaE subunit)
and queG (epoxyqueuosine reductase)
Import this gene and its two neighboring genes into Benchling. List how much
overlap or how many base pairs are in between NNR and its two neighboring genes.
>2 base pairs of overlap between 5' end of queG, and 29 base pairs of overlap with
5' end of tsaE
Look at the protein sequence of NNR. What is the codon and amino acid of residue
210 (full name, no 1 or 3 letter abbreviations)? Include a screen shot showing the
first part of the protein translation here (show at least the first 50 amino
acids).
>amino acid at residue 210 if glycine, gly, G
Design primers to PCR amplify the NNR gene, and aim to generate a PCR product that
is around 1600 bp. Annotate these on your DNA sequence, and import the resulting
sequence as a genbank file. Make sure the file has all three genes and your primers
annotated. Name the file as Lastname_Firstname_PS1_Q1
Q2: Visualizing mammalian gene sequences.
Look up the human UNG gene in Benchling. How many exons does this gene have?
>7 exons
What is the codon and amino acid (full name, no 1 or 3 letter abbreviations) of
residue 178 of this protein?
>Serine, Ser, S
Design primers to PCR amplify exon 5. Force the forward primer to bind across the
exon 4/intron 4 boundary, and force the reverse primer to bind across the intron
5/exon 6 boundary. What will the size of your PCR product be? Annotate both primers
>PCR product is 1452 base pairs
Import the resulting sequence as a genbank file. Be sure your primers are
annotated. Name the file as Lastname_Firstname_PS1_Q2
Q3: Visualizing plasmids.
Look up the pCas9 (plasmid #42876) plasmid in addgene and import into Benchling.
What antibiotic would you use when working with this plasmid, what is the
antibiotic resistance gene annotated as, and what is the promoter annotated as?
>Use Chloramphenicol antibiotic, annotated as CmR with the promoter annotated as
cat promoter
What is the origin of replication of this plasmid? What will the approximate copy
number of the plasmid be (you may have to do some searching to find this – addgene
has some great resources!)?
>The origin of replication is annotated as p15a, which is listed as havbing a copy
number of about 10 - [Link]
Look at the protein sequence of the Cas9 gene. Cas9’s two catalytic residues are at
positions 10 and 840. What are they?
>Position 10 - D or aspartic acid
>position 840 - H or Histidine
Design primers to PCR amplify the Cas9 gene. Import the resulting sequence as a
genbank file. Be sure your primers are annotated. Name the file as
Lastname_Firstname_PS1_Q3