Cloning and Expression of

Thermococcus Kodakarensis Cdc6

Protein in Pichia Pastoris

Yasmin Moez
Institute for Bioscience and Biotechnology Research, 9600
Gudelsky Drive, Rockville, MD 20850
 Abstract
DNA replication is an essential process for all domains of life.
Bacteria, Archaea, and Eukarya utilize DNA replication to duplicate all
genetic information. With the current knowledge of DNA replication, the
Cdc6 protein is hypothesized to be present during the initiation phase of
the replication process in Archaea and Eukarya. Nearly all archeal species
are found to have encode for the Cdc6 protein. The goal of the project is
to purify the Cdc6 protein to examine what exactly its purpose is with
respect to DNA replication. Yeast was used to express the Cdc6 protein
because when Escherechia Coli was used, the protein was not soluble,
therefore yeast was used to express it.
 pPICZA Vector

The enzymes EcoR I and Kpn I were used to cut both pPICZA and Cdc6 protein. The length of the plasmid is
3.3 kb and that of the Cdc6 gene is 1.2 kb, meaning the resulting, ligated plasmid should be 4.5 kb.
 Processes Involved (1)
Polymerase Chain
Reaction (PCR)of Digestion and
Cdc6 gene Purification of
(to amplify) Cdc6 (to cut) Ligation and
Transformation of
Cdc6 and pPICZA
Transformation, (together)
Extraction, and Gel Digestion and
Electrophoresis of Purification of
pPICZα pPICZα (to cut)
(to amplify & confirm)

Extraction,
pPICZα was used Digestion and Gel
repeatedly but the Electrophoresis of
Cdc6 protein was plasmid to confirm
never confirmed to if insert is present
be inserted
 Processes Involved (2)
Same procedure as with Prepare competent
pPICZα, was repeated yeast cells Mix yeast cells
on pPICZA and insertion with linearized
of Cdc6 was confirmed plasmid and
Digestion and spread on plate
Extraction of with Zeocin
pPICZA
(to linearize)

Induce yeast to Confirm insertion

Extract and purify express pPICZA + of pPICZA + Cdc6
Cdc6 protein Cdc6 in yeast

Analyze the Cdc6

protein
 Methods Used
• PCR
• Heat shock Transformation
• DNA Extraction with Qiagen Minispin Kit
• Gel Electrophoresis
• DNA Digestion
• DNA Purification with Qiagen PCR Purification Kit Electroporation Device

• DNA Ligation
• Yeast Transformation by Electroporation

Externally applied electric field increases electric

conductivity and permeability of cell membrane to
introduce something into cells
Transformed bacteria on LB plate + Zeocin plate

Colonies suggest that the Cdc6 protein may have been successfully inserted
because growth occurred in the presence of the antibiotic, Zeocin
Gel confirmation of extracted and digested pPICZA with Cdc6

2 4
3 5
1

The DNA Ladder marks the corresponding length to each band, helping to approximate the length of the
loaded samples. Lanes of extracted pPICZA with Cdc6 (2, 4) show only one band corresponding to the
length of the entire plasmid, 4.5 kb. Lanes of extracted and digested pPICZA with Cdc6 (3, 5) show two
bands, corresponding to the cut plasmid and the inserted DNA, 3.3 kb and 1.2 kb, suggesting the DNA has
been successfully inserted.
 Summary
• Cdc6 protein was repeatedly attempted to be inserted in pPICZα, but was
never confirmed
• pPICZA was used instead and confirmed
• Instead of using E. Coli to express the pPICZA + Cdc6, yeast was used
because in E. Coli, the protein was not soluble
• Transformed yeast has been growing on a selective media compared to a
negative control, which had no yeast growth, leading us to believe that we
have the DNA fragment in the cells
• However, we have yet to confirm whether the insert is present in the yeast
or not.
 Future Work
• Confirm the insert in yeast
• Induce cells to express the Cdc6 protein
• Extract and purify the protein
• Analyze the protein (activity, biochemical characterization)