Construction of a Gene for an Artificial Enzyme
Taylor Keeney, Dr. David Speckhard
Background
DNA has been subjected to controlled protein synthesis by genetically modifying the
organism’s genome. Genetic engineering is a process of isolating a DNA sequence and inserting
it into a specific site within the genome. These different sequences of DNA are transcribed into
specific proteins. Being able to design proteins that have a specific function is an important area
of research for pharmaceutics and medicine. One such area of research is designing a protein
with multiple functions. A current novel idea is to design a protein by fusing two domains
together. Metalloproteins are proteins that are capable of binding metal ions and DNA.
This project will aim to construct a gene for an artificial enzyme which can be used to
study how protein’s structure and function are related. Artificial proteins which bind to metals
and promoter regions of DNA a certain way would have a great influence on selective gene
regulation. If we can construct a metalloprotein, it is one step closer to designing nucleases to
control genes in the cell.
Sonya Franklin, Department of Chemistry at the University of Iowa, studied de novo
metalloprotein design creating a biological motif mimicking lanthanide-binding and a
supersecondary turn structure capable of serving as a hydrolytic cleavage site, named proteins P3
and P4 (1). The design of these proteins consist of helix-turn-helix (HTH) and EF-hand motifs
which have two helices at right angles from one another. P4 contains a Calmodulin loop III and
has α2 and α3 of Engrailed without the β-turn and last turn of α2 compared to the P3 version
engineered by Franklin (1). CD titration studies
showed P3 dimerizes at high concentration whereas P4
does not. Calcium+2 is capable of being bound by EFhand chimeras, so a hydrolytic phosphate cleavage
assay was ran to determine which protein can better
bind metal ions and nucleases. P4 ended up binding at
higher phosphate cleavage rate a higher rate due to it
not dimerizing at high concentrations (1). This was significant to show P4 could be used as a
novel-metalloprotein.
Franklin along with Sunghyuk Lim, later engineered a Calcium Lanthanide binding site
into an already DNA-binding scaffold. Using modular turn-substitution, they were able to
genetically engineer four different proteins based off the sequence of the P4 protein (2). TRP
fluorescence spectroscopy of metal binding affinities and an agarose gel retardation assay
showed two of the proteins resulted could bind both metal ions and circular DNA (2). These
results indicate that a gene can be genetically engineered by adding two domains together and
retaining their function.
Kelli Theisen, Loras College graduate, tried to create the P4 gene using PCR. She failed
to successfully create the P4 gene using PCR possibly due to a mitochondrial insert in the
genetically engineered fragment. Instead she used Xho I and Nde I enzymes to create a double
digested sample from a C-2 plasmid for insertion into DH5α E. Coli cells. Synthesis of the P4
gene was attempted by process of annealing but the plasmid was not successfully created.
Because of this, pET 21a was ordered, isolated, and digested with Nde I and Xho I. The P4 gene
was ligated into the double digested pET21a plasmid but did not produce the correct product.
Due to this outcome, DH5α cells were transformed with various combinations of the annealed
gene. After colonies were grown using the extracted insert and the pET21a cleaved cite, samples
were sequenced in Iowa City. Because the P4 gene was not confirmed by the University of
Iowa, it could not be inserted into cells to create the protein (3).
Due to Kelli’s unsuccessful attempt to create the P4 protein, the objective of her project
will be continued at Loras College. The goal of this project is to genetically engineer DE3 cells
to generate the P4 protein. This will be done by vector insertion. DNA containing a constructed
sequence must be isolated to create an expression vector for the P4 protein. The plasmid must be
purified in order for purchased DNA to be ligated into the cut plasmid. After colonies are grown,
the product will be taken to the University of Iowa to be sequenced. If the sequence is verified
as the P4 gene, DE3 E. Coli cells will be grown for transformation. If the transformed DE3 E.
Coli successfully produces the P4 protein, P4 will be isolated and purified in order to analyze its
hydrolytic activity to cut DNA. This will be done to characterize the protein’s ability to act like
a metalloprotein. These steps described will lead toward supporting that the P4 protein can be
created by vector insertion and has the characteristics consistent with a both homeodomains.
Methods
Plasmid Isolation
A pET21a plasmid was obtained from Dr. David Speckhard. The plasmid was isolated using a
ZymoPure Maxi Prep Kit.
Digestion of pET21a Plasmid
A prepared pET21a plasmid was digested with Nde1, Xho1, and Nco1. A control was ran with 1
uL of BSA, 15 uL of DNA, 2 uL of Buffer #4, and 2 ul of ddH2O. All three enzymes were used
for single cut. The single cut reaction had 1 uL of BSA, 15 uL of DNA, 2 uL of Buffer #4, 1 uL
of ddH2O, and 1 uL of the desired enzyme (Nde1, Xho1, or Nco1). The final reaction was the
double digest. This reaction had 1 uL of BSA, 1 ul Xho1, 1 ul Nde1, 15 uL of DNA, 2 uL of
Buffer 4, ad 1 uL of ddH2O. The samples were vortexed and then centrifuged down. Next, they
were incubated at 37o C for 30 minutes.
Gel Electrophoresis
Each digested sample was analyzed by gel electrophoresis. The gel was prepared using .4 g
agarose dissolved via a microwave in 50 mL 1x TAE buffer. After the solution cooled, 50 uL of
.5 mg/mL ethidium bromide was added to the solution. The solution was poured onto a gel
electrophoresis tray and a lane comb was inserted. The gel was allowed to cool and harden for
approximately 30 minutes. The gel was loaded onto the electrophoresis machine and 1x Tae
buffer was added to cover the gel. 1 uL 10x loading dye to 9 uL of each of the following: control
(no enzyme), single cut Xho1, single cut Nde1, single cut Nco1, double cut (Xho1 and Nde1).
The gel was ran on 60 mA for 45 minutes. The gel was analyzed using UV light.
Gel Extraction
DNA from the single and double digest were excised using a sterile razor blade. The excised gels
were weighed and placed in an Eppendorf tube. For every 100 mg of gel, 300 uL of Buffer QG
was added to the sample. The gel was dissolved for 10 minutes at 50o C. After the gel was
dissolved, 1 volume of isopropanol was added to the sample. The sample was then placed in a
QIAquick spin column and spun in the centrifuge for 1 minute. The flow-through was discarded
and the column was rinsed with 500 uL of Buffer QG. The solution was spun again and flowthrough was discarded. The column was washed with 750 uL of Buffer PE and the flow-through
was discarded. The QIAquick column was then placed in a sterile Eppendorf tube and eluted
with 50 uL of Buffer EB.
Biotek Microplate Reader Gen5 TM
DNA was quantified via UV spectroscopy using Gen5 Take 3. The plates were cleaned with
ethanol and blanked. Once the blank was successful, 2 uL of extracted DNA was loaded on the
sample docks to be read. Readings were took for the single cut Nde1 and Xho1 restriction
endonucleases and for the double digest (Nde1 with Xho1).
Transformation of Dr. Cooper’s cells and Iowa City DNA
D5α cells obtained from Dr. Kate Cooper were thawed by and stored on ice for 10 minutes. 1 uL
of pET21A DNA was added to the cells and stored on ice for 30 minutes. The tubes were then
transferred to a water bath at 42oC for 90 seconds. The tube was then chilled in an ice bath for 2
minutes. 800 UL of SOC medium was added to the tubes and then stored in a water bath for
37oC for 45 minutes. 150 uL of the solution was transferred to a LB plate with kanamycin and
incubated overnight. 150 uL of the solution was also transferred to a LB plate without and
incubated overnight.
Ligation of Single Cut DNA
DNA cut with Nco1 was ligated by mixing 2 mL of ligase buffer, 1 mL of ligase, 3 mL of single
cut DNA, and 12 mL of ddH2O in a sterile Eppendorf tube. The solution was vortexed and
allowed to react at room temperature for 1 hour, then stored in the freezer.
Figures
6 5
4
3
2
1
Figure 1. Digestion of the pET21a plasmid. The plasmid was digested and examined on a 1.2% agarose
gel for 45 minutes at 60 mA (6). The plasmid was cut with Nco1 (2), Nde1 (3), and Xho1 (4). The double
digest consisted of Nde1 and Xho1 enzymes (5). The ladder is in lane 1.
Figure 2. UV spectrum analysis of single digest pET21a plasmid. The single digests of Nde1 and Xho1
were analyzed twice to determine the concentration of DNA in the sample. Nde1 was analyzed in Lane
2 and Xho1 was analyzed in Lane 3.
Figure 3. UV spectrum analysis of double digest pET21a plasmid. The double digest with both Nde1 and
Xho1 was analyzed to determine the concentration of DNA in the sample. The sample was analyzed
twice in Lane 2 and 3.
Figure 4. Transformation of D5α cells with pET30 DNA. D5α cells were transformed with pET30 DNA
and spread out on a kanamycin plate. The plate was left in the incubator overnight.
Discussion:
To understand the P4 protein in a cost effective way, E. Coli was used. The plan is to
design an active form of the protein that can be used to study enzyme kinetics and the hydrolytic
activity of proteins. The design of the P4 protein is one step closer to being able to design novel
metalloproteins with adjustable hydrolytic activity (1,2).
In order to design the P4 protein, a pET21a plasmid was ordered. This was chosen over
the plasmid from pET30 because this plasmid needed Nho1 and Xho1 enzymes for the primers
already ordered. If pET30 was used, an enterokinase would have been needed to cut the histag
off after the protein was already synthesized. pET21a was digested with Nco1, Xho1, and Nde1
enzymes as seen in Fig1. The Nco1 enzyme was only used for procedural purposes to see if any
of the enzymes would cut because Nco1 itself serves no use in the ligation. A control was not
run on this gel but should have shown the DNA in supercoiled form. When the plasmids were
cut with the single enzymes Nco1 (Lane 2), Nde1 (Lane 3), Xho1 (Lane 4), the enzymes
appeared to partially cut the DNA as there was two bands representing the super coiled and
circular DNA, as the cut relieved tension in the DNA. The double digest using Nde1 and Xho1
enzymes appear to have been successful as the double digest caused the DNA to collapse into
linear form and run farther towards the bottom of the gel than either the super coiled or circular
DNA. All bands in the gel (Fig 1) were fairly light so the double cut DNA as well as the single
cut DNA were extracted from the gel so the single cut DNA could be double digested.
The extracted DNA was used first examined under UV spectroscopy to determine if there
was enough amount of DNA in the extracted 50 uL samples. After the tests results proved there
would be a significant amount of DNA, the project could move for towards the goal of ligation.
Because the ligated DNA must be transformed, competent cells must be available and the
transformation procedure must be tested before the ligated pET21a plasmid is ligated. This was a
test to rule out if the competentcy of the cells and efficiency of the transformation procedure if
we would happen to not get colonies when transforming the ligated DNA. First, DE3 cells
obtained from Dr. Fuentes lab in Iowa City were transformed with extracted pET30 DNA.
pET30 is kanamycin resistant so kanamycin plates were used. This resulted in no colonies so the
transformation was tried with pET30 DNA and D5α cells obtained from Dr. Kate Cooper.
Because the transformation resulted in colonies, all that was needed to make sure the double
digest should work would be to make sure the ligation procedure worked. DNA cut with Nde1
was ligated and transformed into D5α cells…..
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Made compotent DE3 cells
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Attempting transformation with ligated DNA – Colonies without transformation
Attempting transformation with pET21a PDZ, cells on Amp plate, cells on No amp plate
to check and see if plates work.
o Lawn on NO Amp (expected), Colonies on Amp plate (Not expected), Nothing on
transformation (Not expected)
Reattempt to see if I mixed up the plates
1. Welch, J. Sirish, M. Lindstrom, K. & Franklin, S. “De Novo Nucleases based on HTH ahd EFHand Chimeras” Inorganic Chem. 40 (2001) 1982-1984
2. Lim, S & Franklin, S. “Engineered lanthanide-binding metallohomeodomains: Designing folded
chimeras by modular turn substitution” Protein Science. 15 (2006) 2159-2165
3. Theisen, K., & D. Speckhard. Synthesis of the P4 gene and insertion into the pET21a
vector. 2010.
4. SIGMA GenElute HP Plasmid Midiprep Kit User Guide. 08/2005
5. QIAGEN Plasmid Purification Handbook. 09/2000