Results Self ligation of the pTRE3G-Thiaminase vector resulted after the attempted ligation of the purified and digested FAK promoter into the purified and digested pTRE3G-Thiaminase vector.

Discussion While the experiment itself follows a protocol that checks for successful intermediate steps through PCR followed by gel electrophoresis analysis, the final ligation still contains the possibility of producing several incorrect products. Self ligation of the pTRE3G-Thiaminase vector was seen in each gene sequence sample taken from the wells that were thought to contain product.

Future Work Future development of this experiment can be carried out using different restriction enzymes and primers designed for them. Instead of using Xho I and Sal I as restriction enzymes, a similar project will be carried out using EcoR I and Sal I which should create more compatible restriction enzyme cuts.

Acknowledgements This project was supported by the Texas A&M University College of Science. I would also like to thank my advisor, Dr. Coran Watanabe, my mentor Brian E. Young, and the other members of the Watanabe group for their continuing support in my endeavors.

Abstract Thiamin, also known as vitamin B1, is an important enzyme cofactor for living cells. Thiaminase, an enzyme that degrades thiamin, can be cytotoxic to cells by causing acute thiamine starvation. In situ expression of thiaminase can be controlled using different gene promoters. Focal adhesion kinase (FAK) promoter is over expressed in many cancer cell types that are prone to metastasis. This experiment describes the isolation of the FAK promoter from human genomic DNA and its ligation upstream of the thiaminase gene. The FAK promoter will be activated in cancerous cells to produce thiaminase in order to achieve selective

destruction.

Introduction There are several steps involved in the isolation of the FAK promoter and its ligation into the pTRE3G-Thiaminase vector. The first is to amplify the genomic DNA template containing the FAK promoter using primers designed to be compatible with the restriction enzymes Xho I and Sal I. These restriction enzymes were specifically chosen because they flank the TRE3G promoter to be digested in the final ligation. The FAK promoter is first ligated into Promega’s pGEM T Easy Vector in order to screen for positive FAK promoter ligation into the plasmid insert. Both the FAK promoter and the pTRE3G-Thiaminase vector are digested with the restriction enzymes, and then the promoter is ligated into the linearized vector.

Isolation of FAK Promoter and Ligation into pTRE3G Vector to Control Thiaminase Production Fred Christian G. Velasquez, Brian E. Young, Dr. Coran Watanabe*

Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012

Cancer Cell

Thiamine

pTRE3G-Thiaminase Vector with FAK Insert

Thiaminase

Methods

Metastatic Cancer Cell Conditions

FAK Promoter

in Genomic Template

Activated to Produce FAK

Protein

Isolated FAK Promoter Ligated into pGEM T Easy Vector

Isolated FAK Promoter by Digestion from T Easy Vector

Digested pTRE3G-Thiaminase Vector

pTRE3G-Thiaminase Vector with FAK Insert

Amplify the genomic DNA containing the

focal adhesion kinase (FAK) promoter

100bp

Ligate the promoter into T Easy Vector then blue-white screen and isolate plasmid DNA

from positive colonies

100bp 1 - 9 neg

Digest the colonies containing plasmid DNA

and digest pTRE3G-Thiaminase to remove PTRE3G promoter and

linearize vector

Purified pTRE3G-Thiaminase

Xho I

Sal I

Xho I Sal I

FAK Promoter

pGEM T

Easy

Vector

Ligate FAK promoter into linearized vector

FAK Promoter

Digested

pTRE3G-

Thiaminase

pTRE3G-Thiaminase Vector

with FAK Insert

1kb 1 3 7 10 100bp

Digested FAK Promoter

TCGAG CAGCT C G

Digested pTRE3G-Thiaminase

TCGAC

GAGCT G C

c