MARQUIS LAB PROJECTS
PROTEIN PROJECTS
1 SEARCHING FOR NOVEL ENZYMES FOR DIPEPTIDE SYNTHESIS ; Gamma-glutamyl transferase is a ubiquitous enzyme and is found to have use in the production of the dipeptide gamma glutamyl cysteine. Currently, the enzyme is sourced from native mammalian tissue. This project will explore alternative native and recombinant methods to generate active enzymes for improved industrial application of this enzyme.
2 RECOMBINANT REDUCTIVE DEHALOGENASES ; Reductive dehalogenases are enzymes involved in the reductive dechlorination of polychlorinated hydrocarbons, such as choloroform. Microbial processes to degrade chlorinated hydrocarbons have been described, however the anaerobic processes in particular are relatively slow, because of low cell densities, slow growth rates and low substrate concentrations. This project will continue our work on developing and evaluating a recombinant version of an enzyme that degrades chloroform.
3 RECOMBINANT SPIDER SILK DEVELOPMENT; Spider major ampullate silk is nature’s toughest fibre. In order to commercialise silks for specialist functions, a recombinant approach has been pursued in bacterial and yeast hosts. The fibres generated from the proteins nonetheless have not performed as well as spider silks on tensile and other mechanical performance tests. New genomic data is now telling us that the best performing spider silks are a unique mix of MaSp1 and MaSp2 and other ampullate proteins (spidroins; (e.g. MaSp3 and 4). Some of these ‘other’ spidroins have only recently been discovered and sequenced. This project will systematically isolate, amplify, and express each of the spidroins in a microbial host. The proteins will then be purified and concentrated before being spun into threads using microfluidic techniques. This work will provide us insights into the mechanisms by which the expression of particular genetic patterns and the subsequent proteins are utilized to produce, both naturally and synthetically, nature’s toughest fibres.
BIOPROCESS DEVELOPMENT PROJECTS
1 MICROFLUIDICS FOR MAMMALIAN CELL BIOPROCESSING; Cultured mammalian cells produce valuable biopharmaceuticals and potentially provide tissues for autologous transplantation therapies. Perfusion processes are potentially useful approaches to generate high cell density cultures, suitable for industry. This project will examine application of unique microfluidic devices developed by Dr. Majid Warkiani at UTS to facilitate rapid cell separation for application in recombinant protein production processes.
2 BIOREACTORS FOR INVESTIGATING REDUCED BIOFILM FORMATION IN MODIFIED PVC; PVC conduits are widely used for the supply of drinking water. Microbial biofilm formation in water conduits can result in a reduction in drinking water quality; hence, there is a desire to reduce the propensity for biofilm formation by developing “self-cleaning” materials that deter biofilms by chemically-mediated nitric oxide release.
This project will examine the use of newly acquired stirred and drip-film bioreactors to establish freshwater biofilm communities on PVC and then undertake biofilm deterrence experiments on novel PVC materials generated by our collaborators.
3 FUNCTIONALISING OUTER MEMBRANE VESICLES FOR RECOMBINANT EXPRESSION OF MEMBRANE-BOUND PROTEINS ; Outer membrane vesicles (OMVs) are “blebbed” from the surface of gram-negative bacteria such as E.coli. Under certain conditions, the rate of blebbing increases. A number of genes have been identified that control blebbing, and hypervesiculating strains have been developed that bleb at higher rates. These hypervesiculating strains provide a potential means to secrete membrane-bound recombinant proteins and create functionalised OMVs with one or more recombinant proteins present in the “blebbed vesicles”. In this project, we are interested in exploiting this property to create functional enzyme cascades in secreted OMVs.
INDUSTRY-LINKED PROJECTS - BONDI BIOWORKS
1 PATHWAY ENGINEERING FOR TERPENE BIOSYNTHESIS; Cultured Terpenes and terpenoids comprise a large group of structurally diverse metabolites, produced predominantly by plants, with applications ranging from pharmaceuticals, agrochemicals, through to flavours and fragrances. Most eubacteria, plants and cyanobacteria share the same metabolic pathway (MEP pathway) supplying the cell with terpenes and terpenoids that fulfill various essential functions in photosynthesis, cellular metabolism, cellular defense, etc.. Besides transcriptional control of pathway gene expression, optimisation of the MEP pathway involves identifying limitations at the protein level. Soluble and functional expression of recombinant enzymes in the MEP pathway are crucial to the creation of core chassis strains.
In collaboration with researchers at Bondi, this project involves characterisation and optimisation of terpene/terpenoid synthase expression using a range of biochemical characterisation methods. Comparing expression levels of enzymes from different genetic backgrounds, enzyme fusions and solubility-tagged enzymes allows for an understanding in future rational enzyme engineering approaches and pathway optimisation. The project combines techniques in protein biochemistry (SDS-PAGE, Western Blot, chromatography techniques) and can be further extended to develop novel techniques to increase solubility and function of crucial enzymes involved in terpene synthesis.