BioE 498 Lab Exercise 1
Downloading and learning A Plasmid Editor (APE)
1. Download the APE program from
http://www.biology.utah.edu/jorgensen/wayned/ape/.
2. Download the three APE files from the email: E0240 in pSB1A2, J04450 in
pSB1A2, and R0011 in pSB1A2.
3. Open each file in APE.
4. For each file, you should see sequence data for the top DNA sequence strand in
the 5’ to 3’ direction with BioBrick parts and primer sequences highlighted. Only
the primer sequence that is homologous to the template DNA is highlighted.
Scroll your mouse over a particular sequence and the part or primer sequence
name will display above. Note that there will be multiple displays for some
sequences when there is overlap.
5. The part sequences going from top to bottom include the suffix (with particular
restriction enzyme sites), ColE1 origin of replication sequence, ampicillin
resistance cassette, prefix (with particular restriction enzyme sites), and then the
part (R0011, E0240, or J04450). Then after the part starts the suffix again since
this is a circular plasmid displayed in linear form. This basically means the part is
in between the prefix and suffix.
6. The primer sequences include the P1-P6 primers that overlap with some of the
parts. The P6 primer is displayed twice on the R0011 in pSB1A2 file, ignore
the one on top, only the one on the bottom is correct. I can’t get rid of that
for some reason. The P3 primer is VF2 and the P4 primer is VR. Both of these
are displayed.
7. Based on the Lab Introduction file, first lab manual, and APE files, you should be
able to answer the following questions.
Questions
1. Do the forward or reverse primers match the template DNA (plasmid) in the 5’ to
3’ direction?
2. Do the forward or reverse primers match the reverse complement of the template
DNA in the 5’ to 3’ direction?
3. After identifying all the primer sequences, what are the predicted PCR product
sizes for each PCR reaction you ran?
4. What are the exact portions of the GFP and RFP P1 and P6 sequences that
have homology (are reverse complements of each other)?
5. What end of the primer do you put a sequence that is non-homologous to the
template DNA, the 5’ or 3’ end?
6. What primer sequences would you order to re-engineer the GFP circuit to have a
B0034 RBS instead of B0032 RBS? You would still want to use the R-vector and
GFP-insert plasmids as template DNA. Another way to phrase the question: the
lab protocol uses P1 and P2 primers with the GFP-insert and the P5 and P6
primers with the R-vector. Which would be the same and which would be
different? What are the exact sequences for the different primers?
Email your answers to: sleight@u.washington.edu and I’ll let you know if you got
them right!