CHEM 4700
Homework 4
The Experiment:
The lab that you are working in studies the protein MAPK1 or mitogen activated protein kinase 1. This
protein functions in a variety of pathways and you would like to be able to control the expression of the
protein. Therefore, you have selected the pTRE-Dual2 plasmid as part of your experiment. You need to
produce a Serial Cloner file that contains the MAPK1 coding sequence in the appropriate spot in the
pTRE-Dual2 plasmid. What restriction sites COULD you use? What restriction sites CAN’T you use?
What restriction site DID you use, and did you need to add any spacer nucleotides to adjust the reading
frame? In addition, you need to explain what each portion of the pTRE-Dual2 plasmid does, and what
other plasmid you would need to make everything work properly. Be very clear about your WHY you
are choosing the reagents you do. To facilitate easy visualization of the plasmid, please color-code the
plasmid in the following way:
PTight – Blue
mCherry – Red
IRES2 – Blue
SV40 polyA – Yellow
ColIE1 ori – Grey
Ampr – Purple
MAPK1 - Green
Please type your answers to the questions written above, and submit your Serial Cloner file
electronically. The assignment is due October 28, 2013.
Rubric:
Content – 25 points
- Correct labeling (2 pts)
- Correct definitions (6 pts)
- Restriction analysis (4 pts)
- Correct Cloning (10 pts)
- Other reagents (3 pts)