Lab Report 2
by Guiherme and Siyang
The objective of the second week is to make us familiar with DNA sciences, which are
useful tools for modern biological sciences. There are two main purpose of this lab.
The first one is to clone lacZ gene from E. Coli into plasmid pZE21. The second purpose
is to measure single cell gene expression using GFP as reporter. Also along the way, we
digested -DNA HindIII fragments with EcoRI and use it as DNA ladder.
For gene cloning, we first need to extract the lacZ gene (our insert) from a wild type of E.
coli (MG1655, GenBank U00096) with PCR. For some unknown reason, the PCR
using E. Coli cells does not produce the desired DNA fragment on gel electrophoresis.
Instead, we cut out the PCR product using plasmid pZE21-lacZ as template. After
purification, we put it into pZE21 vector. pZE21 vector (2948 bp) has moderate copy
numbers of 60-70 copies. It has origin of replication colE1, a resistance marker
(antibiotic resistance) of Kanamycin, and a regulatory unit (promoter), PLtetO-1, which is
repressed by the Tet repressor and induced by tetracycline (ATC). Then we transform
the plasmid we made into E. Coli DH5 cells which do not have lac repressor. Also we
set up transformations of killer cut to eliminate the effects of relegation and compare
without killer cut. “Killer cut” is a restriction digest of the ligation mix using a
restriction enzyme that cuts within the starting vector but does not cut the ligated product.
This will select against non-recombinants. The rationale for killer cuts is that any
contamination with religated starting vector or intact starting vector will be linearized and
thus will transform bacteria much less efficiently than the circular intermolecular ligation
product.
In gene expression measurement, we transform plasmid pZE21-GFP into E. Coli
MC4100Z1 cells. MC4100Z1 cells have plasmid for Kanamycin resistance. Then we
can measure and compare the induction with ATC. The negative controls are
MC4100Z1 which do not have lacZYA but have lacI, tetR and araC, and MC4100Z1pZE21-LacI. The later one is a better control because it has plasmid pZE21-LacI, which
is expected to have the same copy number as MC4100Z1-pZE21-GFP, thus better for
cancelling auto fluorescence. The positive control is MC4100-pZE21-GFP, which
doesn’t have any repression system for GFP expression. From our results, clearly the
GFP expression can be induced by ATC. The fold change increases with ATC
concentration approximately exponentially.
We also performed digestion of lambda DNA and analyzed the fragment size. We start
with lambda DNA HindIII digest from Invitrogen. We digested it with EcoRI and run
electrophoresis of the DNA before and after digest side by side.
From gel
electrophoresis, the 4,361bp fragment is almost missing even before EcoRI digestion.
This fragment is from the 3’end. It’s likely that we don’t have this fragment in the
HindIII digest from Invitrogen. Other fragments corresponds well before and after
EcoRI digestion.