Graduation Date

Spring 2021

Document Type



Master of Science degree with a major in Biology

Committee Chair Name

Dr. Jianmin Zhong

Committee Chair Affiliation

HSU Faculty or Staff

Second Committee Member Name

Dr. Mark S. Wilson

Second Committee Member Affiliation

HSU Faculty or Staff

Third Committee Member Name

Dr. Amy Sprowles

Third Committee Member Affiliation

HSU Faculty or Staff

Fourth Committee Member Name

Dr. Heidi Rutschow


E. coli, qPCR, Plasmid, Cloning, Subcloning, Phenotype

Subject Categories



Nutritive symbiosis between bacteria and ticks is observed across a range of microbes and hosts, however, little characterization on the molecular components responsible for these symbioses has been done. Previous studies in our lab demonstrated that Rickettsia monacensis str. Humboldt (RMSH) can synthesize folate de novo via folA, folC, folE, folKP, and ptpS pathway. In this study expression of RMSH folate genes within a folate gene mutant E. coli K-12 strain BW25113 was used to functionally characterize RMSH folate genes in vivo. RMSH folate genes were subcloned into TransBac vector and transformed into folA, folC, folE or folK knockout mutant E. coli K-12 strain BW25113. Each mutant containing a RMSH subclone and a pFE604 clone of the knocked-out folate gene was cured of pFE604. Curing of folA mutant was successful using acridine orange and 43.5ºC but curing folE and folK mutants was only achieved after supplementing plasmid curing media with folate end-products. Preliminary plasmid curing assay showed curing efficiency of folA mutant at 100%, whereas curing efficiencies for other mutants was 0% to 10%. Functional complementation was assessed by growth phenotype on minimal media with and without IPTG between RMSH folA and E. coli folA as well as RMSH folC and E. coli folC gene pairs. Large and homogenous wild-type colony growth was observed for both assayed gene pairs on minimal media with IPTG, and lack of growth or pin-point growth without IPTG. This study provides evidence substantiating the in vivo functionality of RMSH folate genes in producing functional gene products for folate biosynthesis.

Citation Style


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