Title

Using Golden Gate Assembly to Rapidly Create Genetic Constructs for Transgenic Plants and Promoter Analysis

Date of Award

Spring 2015

Document Type

Thesis

Department

Biology

First Reader

Dr. Nathan Reyna

Second Reader

Dr. Ruth Plymale

Third Reader

Dr. Kevin Cornelius

Abstract

Golden Gate Assembly, a genetic assembly method in which the ligation and digestion of oligonucleotides occurs in a single step, can be used for an array of purposes. In order to test and take advantage of this assembly method our lab selected three experiments in which it could be used. These included an oxidative stress sensitive green florescent protein, a plant specific form of Golden Gate Assembly coined "Green Gate Assembly," and a viral promoter verification system called "pClone Red."

Arabidopsis (Arabidopsis thaliana) plants were analyzed for the presence of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]). Blue LED lights with a filter were used to screen plants for GFP. Plants were screened using a non-sterile sand and antibiotic method in order to increase yield of plants containing gene of interest.

Two constructs were created to test the "Green Gate" method, an adaption of the "Golden Gate" method specifically for plant use. This method utilizes highly specific 4 base pair overhangs and a common type IIs restriction site in order to anneal multiple parts into one vector at once. A construct to transform future plants was created using the "Green Gate" method.

A bacteriophage named "Corndog" was analyzed at the 2014 HHMI conference for possible promoters. One of the suspected promoters was created for testing. The promoter was created with specific overhangs so it could be placed in vectors with the "Golden Gate" method. After annealing, the promoter sequence was put into a pClone Red destination vector. The plasmid was then transformed into Escherichia coli cells and analyzed to confirm a working promoter.

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