1. The researcher performed site-directed mutagenesis on T7 DNA polymerase to create a cysteine-free variant, allowing for fluorescent labeling of just the exonuclease domain. 2. They mutated a cysteine codon on the finger domain to a glutamic acid codon. 3. Sequencing of the mutated plasmid DNA from transformed bacterial colonies confirmed the desired mutation, showing the mutagenesis was successful.

1. Mutagenesis of a T7 DNA Polymerase to a Cys-Free Variant
Karen Ayoub
Center for Integrative Proteomics Research at Rutgers University
Background
T7 DNA Polymerase is a high fidelity protein, meaning it is very accurate in
replicating DNA. It not only initially rejects incorrect base pairs, but it also
excises and replaces an unrecognized incorrect nucleoside triphosphate
(dNTP) at the DNA terminus (see Figure 1).
This protein is important because of its fidelity. Researchers hope to
replicate some of its functions in an artificially engineered protein that
could possibly benefit humans. Another potential advantage of studying
this protein is improving DNA sequencing and complementary DNA (cDNA)
synthesis, a process that already utilizes T7 DNA Polymerase. cDNA can be
used in the cloning of eukaryotic genes in prokaryotes.
Exonuclease Site
Palm Domain
dNTP excised from
DNA
DNA
Poly Site
Figure 1: How T7 DNA Polymerase Operates
The long-term experiment in my lab concerns the structure and function of
the T7 DNA Polymerase, specifically, that of its parts. Using a fluorescent
microscope, the research team aims to observe the motion of the protein,
including the following three:
1. Translocation (movement) of the protein along the DNA strand
2. Opening/closing of the finger domain during the binding of new dNTP to
the terminus (end) of the DNA strand
3. Interaction between DNA terminus and exonuclease domain where dNTP
is excised
The focus of my work concerned the third type of motion. I performed a site-
directed mutagenesis that will allow observation of the exonuclease domain.
By using a fluorescent dye, which binds to the cystine amino acid, as a marker
on the exonuclease domain, the exonuclease domain can be monitored.
However, there is also a cystine on the finger domain of the protein, which
means that the dye could bind to it as well, rendering it difficult to distinguish
between movement of the finger and exonuclease domains (see Figure 2).
Experimental Design
I performed mutagenesis which resulted in the creation of an artificial plasmid
(approximately 4.8 kilobase pairs in length) that contains the protein T7 DNA
Polymerase, without the cystine amino acid on the finger domain (see Figure 3).
After the mutation, the DNA sequence at the position of base 1541 to base 1543
should be GAA, which codes for glutamic acid (instead of TGC, which codes for
cystine). I worked with two types of protein, exo plus (+), in which the
exonuclease domain is functioning, and exo minus (-), in which the exonuclease
domain is dormant. Both types will be used for further studies in the experiment.
Exonuclease Site
Palm Domain
Poly
Site
Marker bound to Cys on Finger Domain
Marker bound to
Cys on
Exonuclease Site
Figure 2: T7 Before Mutagenesis
Exonuclease Site
Palm Domain
Poly
Site
No Cys on Finger
Domain
Marker bound to
Cys only on
Exonuclease Site
Figure 3: T7 After Mutagenesis 3. Now that the desired mutant plasmid is isolated, it can be inserted into
ultracompetent cells (that easily accept new DNA). The cells undergo
both cold and heat shocks, which stress the cells and allow their DNA to
open and accept the mutant DNA. The cells are then added to a growth
medium (NZY+), and then placed on agar plates containing ampicillin,
where they can grow overnight. (Results form my experiment: cells
grew, meaning the mutation was succesful, see Figure 4).
Isolating DNA from the cells
To sequence the DNA, it must be isolated from its cells. There are a few steps that
must take place after the cells grow in the agar plate. I used the QIAprep Spin
Miniprep Kit (QIAGEN).
1. A cell colony taken from the agar plate is placed in a tube with LB broth (see
Figure 5) and incubated overnight.
2. The precipitant (cells) is separated from the fluid, which is disposed of.
3. Buffers are added to the cells which perform several functions in isolating the
DNA. The cells (in a spin column, see Figure 6) are washed with each buffer
when placed in a microcentrifuge, and the flow-through is disposed of.
• Cell lysis using detergents
• Absorption or binding of the DNA to silica in presence of high salt
concentrations in column
• Removal of proteins, polysaccharides, and salts
• Separation of genomic DNA from plasmid DNA
• Washing and drying plasmid DNA
• Maintaining pH
Experimental Design (Continued)
Outline of Procedure
• Mutagenesis to Cys-free variant
• Growing mutated bacteria on agar plates
• Growing cell colonies in LB broth (growth media)
• DNA miniprep
• Preparing DNA for sequencing (addition of primers)
• Sequencing of mutated DNA
How Does Mutagenesis Work?
Acknowledgements
• Liberty Science Center
• Partners in Science Program & Staff
• Rutgers University & CIPR
• Dr. Sanghyuk Lee (mentor)
• Matthew Putnins (lab assistant)
Figure 4: Ampicillin-
Resistant Mutant Bacteria
How Does Mutagenesis Work? (Continued)
Mutagenesis is the process of altering an organism’s genes by gradually introducing
new genetic material. Mutagenesis involves several steps. In this experiment, I used
the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies).
1. The first step involves denaturing the original DNA template, adding the primers
with the desired mutation (ampicillin resistance), and annealing both of them
(reforming the double-stranded nucleotide). Then a polymerase chain reaction
(PCR) is performed, which amplifies a portion of mutated DNA.
2. After the PCR is complete, there will be some unmutated DNA, known as the
methylated parental DNA, as well as the mutated, unmethylated non-parental
DNA. An enzyme is used to digest the unmutated parental DNA.
Figure 6:
Spin
Column
Figure 5: LB
Broth
Results: Sequencing and Mutagenesis
Primers, which are short strands of DNA, must be aligned with the DNA to be sequenced.
One of the 7 primers was defunct, and the sequence for the corresponding portion of DNA
could not be derived. However, I was still able to see evidence of successful mutagenesis.
The expected mutation in the sequence occurred (GAA instead of TGC) in several of the
colonies.