Abdullah Hassan Bu Khamsin
Dhahran Ahliyya School, Dammam, SAUDI ARABIA
Statistics show that by 2050, the food production capacity needs to be doubled to meet the rising demands. Genome engineering can be used as a platform for increasing crop yields to meet future demands by inducing stress-tolerant crops. Transcription activator-like effectors (TALEs) are unique proteins that can be used as customizable DNA binding modules which bind to any user-defined sequences. TALE proteins have been used to generate transcriptional activators and chimeric nucleases for transcriptional modulation or genome editing applications. While the construction of tailored TALEs is vital for many biotechnological applications, assembling TALEs remains difficult due to the complex nature of their nearly identical tandem-repeats. Thus, this project utilizes a straight-forward sequential restriction-ligation step which has been tested in vivo to evaluate its efficiency for tailoring TALE transcription factors (TALE-TFs). This was done by relying on a construction library that contains all of the possible combinations of Repeat Variable Di-Residues (RVDs) in pUCMCS vectors. Five type II restriction enzymes and T4 ligase were then used to convert the tailored construct coupled with the scaffold (dHax3) in an expression vector. This resulted in concentrations of the RVD fragments which ranged from 815 Ng/μl to 7.8 Ng/μl. More importantly, designer TALEs (dTALEs) construction was facilitated in 9.5 hours and was validated using the functional testing findings through the transient expression assays of uidA gene in tobacco leaves. This will pave the way for the implementation of TALEs in genome engineering for a variety of applications in crops. Awards won at the 2013 ISEF Second Award of $1,500 - Plant Sciences