Date of Award

5-2022

Degree Type

Thesis

Degree Name

Departmental Honors

Department

Biology

Abstract

Cloning is an important technique in functional genomics, particularly genetics and molecular biology. Cloned genes can be used to facilitate protein expression; this is an essential step in understanding gene function. In traditional cloning, the gene of interest is amplified and tagged with a restriction enzyme sequence and ligated to the digested open vector. However, the GATEWAY™ Cloning Technology provides a speedy and more efficient route to insert the gene of interest in the desired vector, known as Destination vectors or Expression vectors, using site-specific recombination sites and recombinase enzymes.1 Once DNA segments from the gene of interest are cloned in the Entry vector (DONR Vector) by site-specific recombination, the vectors can be used for research. This powerful system can easily transfer one or more DNA sequences cloned in one Entry vector into multiple vectors in parallel reactions while maintaining orientation and reading frame, which are clear benefits over the restriction enzyme cloning method (see Figure1).2

Entry and Destination vectors used in the Gateway cloning system require storage by transforming them into Escherichia coli strain DB3.1. They can replicate in this particular strain because it is immune to the effects of the ccdB gene carried in the Gateway cassette; the cloning strain is not resistant to the ccdB gene, so the empty vector does not survive in the cloning strain. The ccdB gene cassette is replaced by the gene of interest (GOI) in the recombination step. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids (Entry Vector) will consequently allow the growth of standard cloning strains of E. coli (e.g., DH5α).

Therefore, after making new Gateway plasmids stocks, the vector's ccdB gene mutants must be tested. This process involves extracting the plasmid (vector) from a single DB3.1 bacterial colony grown in LB media and transforming the plasmid into both DB3.1 and the preferred cloning strain of E. coli DH5α in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (i.e., no or few colonies are obtained after transformation) should be used in Gateway cloning reactions.3 This process is called propagating gateway vectors. In this study, we standardized protocol to propagate two Entry vectors (pDONR201 (Kanamycin resistant)) and pDONR 207 (Gentamycin resistant)) and two destination vectors (pMDC32 (Overexpression) and pMDC107 (GFP Tag expression)); both are kanamycin resistant. This standardized protocol will be used in future laboratory work to produce a consistent method of Gateway vector propagation specific to laboratory-specific needs.

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Faculty Mentor

Amita Kaundal

Departmental Honors Advisor

Jennifer Burbank