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dc.contributor.authorFryer, Thomas
dc.date.accessioned2022-04-19T13:39:58Z
dc.date.available2022-04-19T13:39:58Z
dc.date.submitted2019-09-30
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/336187
dc.description.abstractThis work establishes a new genotype:phenotype linked display platform: Polyacrylamide Hydrogel bead Display (PHD). Four core elements have been designed into it; compatibility with ultra-high throughput microfluidic workflows, stability of genotype linkage, stability and control of protein display, and compatibility with a diverse range of assays (affinity, catalytic, cell-based) and displayed proteins (scFvs, DARPins, antimicrobial peptides, enzymes). The first chapter presents the development of polyacrylamide hydrogel bead display. To enable protein display on polyacrylamide beads a new molecule is made, methacrylate-PEG-benzyl guanine, which is co-polymerised into the polyacrylamide hydrogel via the methacrylate group. The benzyl guanine group can form a covalent bond with the SNAP-tag. SNAP-tag can be expressed as a fusion to proteins of interest, thus enabling covalent phenotype linkage to beads. Proteins can be displayed at up to 2.8 x 108 molecules per 20 micron bead, and still be detected at only 5000 molecules per bead. Acrydite- modified oligonucleotides can also be co-polymerised into the hydrogel beads, enabling PCR to be used for genotype linkage to the beads. Emulsion in vitro transcription and translation (IVTT) can then be used to monoclonally express the encoded protein, which is then captured on the encoding bead in the droplet, thus establishing on bead genotype:phenotype linkage. The second chapter focusses on the development of library-scale polyacrylamide hydrogel bead display in the context of improving a potential cancer therapeutic agent; a pro-apoptotic, anti-TRAIL-R1 scFv. Emulsion PCR was found to be an unsuitable library amplification technology, and instead amplification of single DNA molecules in microfluidic droplets is achieved by emulsion RCA. Hydrogel bead formation is achieved by “pico-injecting” a polyacrylamide hydrogel mix into each individual droplet, and allowing polymerisation to occur. The polymerised hydrogel beads, functionalised with both genotype and benzyl-guanine, are recovered and used in an emulsion IVTT reaction. The hydrogel beads can be packed on chip, enabling single bead per droplet encapsulation efficiencies far in excess of the Poisson distribution. The in vitro expression of the pro-apoptopic scFv molecules was found to be low yield, limiting its use for cell-based functional screens, thus an alternative protein scaffold, DARPin, was tested and shown to express at high yield in IVTT. Concomitantly it was found that multimerization of the agonistic molecule improves the induction of apoptosis significantly. To enable screening of libraries at desired multimeric states a display construct was made, SNAP- PhoCl-SpyCatcher 3x, that would trimerise library members when expressed as a fusion to SpyTag. Trimerised library members can then be released from bead in droplet through photocleavage of the PhoCl protein. The third chapter focuses on an alternative library construction technique termed splinted ligation. Splinted ligation is a combinatorial technique that achieves diversity through iteration rather than compartmentalisation. Notably combinatorial library construction achieves 100% of beads with a monoclonal genotype. A small test library is created of an antimicrobial peptide LL37, and successful in vitro expression and display confirmed by antibody staining and FACS analysis. Ultra-high throughput compatible bacteriolytic assays based upon either fluorescence or absorbance are shown.
dc.description.sponsorshipBBSRC MedImmune
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectSynthetic biology
dc.subjectBiochemistry
dc.subjectDIrected evolution
dc.subjectMicrofluidics
dc.titlePolyacrylamide hydrogel bead display for ultra-high throughput screening and evolution of therapeutic proteins
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-04-19T07:23:40Z
dc.identifier.doi10.17863/CAM.83612
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
rioxxterms.typeThesis
dc.publisher.collegeHomerton
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (1651279)
pubs.funder-project-idBBSRC (1651279)
cam.supervisorHollfelder, Florian
cam.supervisor.orcidHollfelder, Florian [0000-0002-1367-6312]
cam.depositDate2022-04-19
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement


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