3Jobie Crear

Partners: Erin, Aida, Proud

TA: Anna
Manipulating Bacterial DNA
Intro: When scientists are studying DNA, one of their quicker and more
indirect methods of determining whats inside that DNA is by utilizing
the phenotype of an organisms DNA and examining it visually. Some
variations of certain DNA show up as different colors than other
variations and this can help scientists determine which variation of
DNA a certain organism has. In this lab, we are focusing on E-coli
plasmid DNA which are different from their regular DNA chromosome.
This Dna exist seperateley off on its own in the cell. The plasmid we
will focus on is PUC18 which contains antibiotic resistance genes. We
will try to determine which variation of this plasmid our different e-coli
cells have: will they have foreign additions in their plasmids? Through
isolating the plasmids from the cells we are testing, we can then place
them in gels and do electrophoresis in order to determine the
genotype. In the second part of the lab, we can also use phenotype to
predict the genotype of the DNA by using a process known as
transformation which will allow us to transfer the various plasmids into
a host chromosome inside a host cell. WE can then put these cells in
conditions that they may or may not have developed resistance to
such as a dish with ampicillin in it and a dish with Xgal to see if they
still grow even with these normally fatal conditions. We will even feed
them a nutrient rich substance known as Agar to help facilitate their
growth so that we can more quickly see where cells are growing and
where theyre not. After seeing the cells in their various dishes for
some time, we will be able to see the cells with the resistance genes
prospering in the enviornments they have adapated too and they
should look visually/color different.1
Methods: The procedure on pages 56-58 and 60-61 of the lab manual
were followed exactly as described2
Results:

1 Murolo. Genetic Engineering. In Introductory Biology Lab manual.

Wesleyan, 2014
2 Murolo. Genetic Engineering. In Introductory Biology Lab manual.
Wesleyan, 2014

No Resistance
Natural Resistance (A)
Foreign Resistance (B)

No growth of cells
Growth of blue cells due to
Xgal and resistance to
Ampicillin
Growth of white cells due to
resistance of Ampicillin but

inability to digest Xgal

Discussion:
A:
B: The transformation results are important because they visually show
us what type of cell prospered in each. In the empty sample/Sample C
must be the cells with the blank locus as they didnt prosper at all
since the cell needs the pUC18 plasmid which contains the enzyme
beta-lactamase to fight off the ampicillin thats present in each of
these containers. The Top left/white one/B is the E.coli with the foreign
dna in its polylinker as its unable to produce b-galactosidase and
consequentially unable to digests the Xgal which would turn it from
white to blue. It still prospered though as you can see the many white
cells so it does have ampicillin resistance. The top right/A is the e.coli
with natural ampicillin resistance as it not only thrived in an ampicillin
heavy environment, its B-galactosidase gene was still on to allow it to
digest the Xgal and turn blue.
Sources of Error: If the clone/host plasmids dont take up the DNA,
they wont reflect the dna theyre given at all. We werent able to see
our groups bands clearly because we injected our cells into the gel too
rough causing them to shoot out of their wells. The digestion of the
plasmid can not be complete
Real Life application: If you wanted to make a biological weapon
from bacteria, all one would need to is take a host/clone cell, and inject
it with even more antibiotic resistance genes than we did.
References
1. Murolo. Genetic Engineering. In Introductory Biology Lab manual.
Wesleyan, 2014
2. http://en.wikipedia.org/wiki/Multiple_cloning_site