Investigation into the HutD Protein within the Histidine Utilization (hut) Pathway in Pseudomonas putida
Open Access
- Author:
- Guzman, Nicholas
- Area of Honors:
- Biochemistry and Molecular Biology
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Joseph C. Reese, Thesis Supervisor
Allen T Phillips, Thesis Honors Advisor - Keywords:
- Histidine
Histidine Utilization Pathway (Hut)
HutC
HutD
repressor
promoter
operator
beta-galactosidase
pET vectors
Protein Overexpression
SDS-PAGE
Nickel-IMAC
Molecular Cloning - Abstract:
- The histidine utilization (Hut) pathway confers the ability of an organism to degrade the alpha-amino acid histidine into carbon and nitrogen. While the exact function of most genes of the hut system has been characterized, nothing is known about the role of hutD, a gene co- transcribed with the well characterized repressor gene of the system, hutC. In this thesis, the pMS1 plasmid was first analyzed in an effort to overexpress His6-tagged HutD protein. Work was successfully conducted using cut and paste cloning, agarose gel electrophoresis and Sanger- sequencing to create and confirm the pNG1 plasmid, which was shown to be capable of overexpressing N-terminally His6-tagged HutD when induced with 0.2% isopropyl ß-D-1- thiogalactopyranoside (IPTG). As well, His6-tagged HutD protein along with His6-tagged HutC repressor protein were successfully overexpressed and purified by Nickel-IMAC. These results will allow the future use of an in-vitro electrophoretic mobility shift assay (EMSA) to determine physical binding characteristics of HutC and HutD proteins in the presence of hut operator regions and urocanate. Also, work was conducted to analyze and improve the functionality of an in-vivo beta-galactosidase assay designed to determine the effects, if any, of the HutD protein on mRNA transcription from the hutG promoter. Results were obtained that concluded that the assay is functional; however, results did not show any differences caused by repression/induction using the PBAD promoter. Therefore, work was concluded with redesigning the pJM1 and pJM2 plasmids with the aim to create two new plasmids, pNG2 and pNG3, that are capable of overexpressing HutC or HutCD, respectively, by using the pBAD43 vector. This vector, with a different origin of replication than in the pMLB1034-derived plasmid vector used to assay beta- galactosidase, will allow for insertion and functionality of both vectors in-vivo in an E. coli host.