Notebook

We've been working hard inside and outside of the lab for around four months, and whilst it has been challenging, we've had a lot of fun! Read more about it below.

Nanobody surface display
  • Parts design of nanobodies and Neae
fuGFP-CBD
  • Parts design of fuGFP-CBD
HP and Modelling
  • Brainstormed session of what we would like to do for the Tahgara Winter programme
  • Delegated tasks and set deadlines to different members
Nanobody surface display
  • Ordered parts and associated primers
fuGFP-CBD
  • Ordered parts and associated primers
HP and Modelling
Nanobody surface display
  • -
fuGFP-CBD
  • -
HP and Modelling
  • Had a practice run and ran the Tahgara Winter programme at our lab
  • The structure of nanobody H and sfGFP successfully modelled in OpenMM
Nanobody surface display
  • gBlocks of parts arrive
fuGFP-CBD
  • gBlocks of parts arrive
HP and Modelling
  • -
Nanobody surface display
  • Inserted Neae surface display construct into TOP10 E. coli and induced with X-gal. Notably, there were few blue colonies
  • Performed colony PCR on 2 colonies with Neae
  • Troubleshot lac expression since there were few blue colonies. Maybe due to low copy number plasmid
fuGFP-CBD
  • Inoculated plates with transformed TOP10 cells with the 4 fuGFP-CBD constructs (CBDclos, CBDcex, CBDcipA, CBDcenA) inserted into pUS250
  • Performed colony PCR
  • Resolved issues which arose with transfecting cells with pUS250
  • Induced cells with cumate and measured fluorescence
HP and Modelling
  • Discussed what needs to be done and set deadlines for the faculty of science stall for University of Sydney’s welcome week
  • Docking of sfGFP and nanobody H attempted in OpenMM, with little success
Nanobody surface display
  • Cloned and transformed the 5 nanobodies into TOP10 E. coli
  • Performed PCR on both the nanobodies and neae construct g-blocks to use product in further experiments
fuGFP-CBD
  • Purified fuGFP-CBD samples and ran SDS-Page of 6 fuGFP-CBD samples including flow through and washes
  • Performed another 2 SDS-PAGEs and fluorescence plate readings to test CBD construct binding to filter paper discs and cotton balls
HP and Modelling
  • Ran faculty of science stall for two days
  • Attended information session for science week stall at the Australian museum
Nanobody surface display
  • Performed colony PCR on nanobody cloning/transformation products
fuGFP-CBD
  • Ran SDS-PAGE and fluorescence plate readings to compare different induction concentrations using cumate
  • Repeated assay using cotton and compared induced and uninduced samples of the 4 fuGFP-CBD constructs
  • Determined optimal induction time by running a time course assay
HP and Modelling
  • After weeks of trying, OpenMM abandoned as a tool to model the docking of GFPs and nanobodies, owing to a lack of success and an overabundance of errors
Nanobody surface display
  • Tested nanobody binding to sfGFP and fuGFP
fuGFP-CBD
  • Ran SDS-PAGE of both induced and uninduced of 4 fuGFP-CBDs tested before, including the insoluble fraction for fuGFP-CBDcipA (brightest sample)
  • fuGFP-CBD samples need linker as most of the construct was in the insoluble fraction
HP and Modelling
  • Ran science week stall at the Australian museum
  • LightDock, an alternative molecular dynamics simulation tool, explored as a successor to OpenMM
Nanobody surface display
  • First half of DNA shuffling protocol (digest, and reassembly) on five nanobody gBlocks
fuGFP-CBD
  • Amplified and ran SDS-PAGE of fuGFP-linker-CBDs (newly ordered constructs)
  • Performed golden gate cloning and colony PCR of fuGFP-linker-CBD constructs
HP and Modelling
  • Meeting with Groningen iGEM team
Nanobody surface display
  • Tested nanobody and neae surface display binding with purified GFP
  • Amplification of shuffled products via PCR
fuGFP-CBD
  • Ran SDS-PAGEs and fluorescence plates of fuGFP-linker-CBDs of induced and uninduced samples including the lysate, whole cell, and insoluble fraction
  • Repeated above assay comparing filter paper discs, cotton, and microcrystalline cellulose
HP and Modelling
  • Meeting with Groningen iGEM team
Nanobody surface display
  • Repeat of PCR amplification and electrophoresis gel of shuffled products
fuGFP-CBD
  • Characterised fuGFP-linker-fuGFP binding to cellulose
  • Cloned and ran colony PCR of fuGFP-linkerCBDs into pET28ct and transformed BL-21(DE3) cells as fluorescence was weak using pUS250
  • Ran SDS-PAGE of fuGFP-linker-CBDcipA samples (brightest fluorescence) binding to microcrystalline cellulose
  • Used microcrystalline cellulose from now on
HP and Modelling
  • Meeting with Groningen iGEM team
  • The first successful docking of sfGFP and nanobody H completed in LightDock
Nanobody surface display
  • Transformed shuffled products and plated on X-GAL and IPTG
  • Cloned shuffled products into pUS250-Neae plasmid
fuGFP-CBD
  • Ran fluorescence plate of 4 fuGFP-linker-CBD constructs using different types of buffers to determine the optimal condition
  • Use NT from now on
HP and Modelling
  • Docking of all combinations of GFPs and nanobodies commences, with significant runtimes
Nanobody surface display
  • Screening of shuffled library lysate by washing with PBS and running a fluorescence plate
  • Performed colony PCR of colonies with the shuffled products
  • Tested GFP binding with the shuffled products
fuGFP-CBD
  • Ran fluorescence assay, comparing fuGFP-CBDs and fuGFP-linker-CBDs
  • Also tested different elution conditions using fuGFP-linker-CBDcipA
  • Further testing using glucose, maltose, and glycerol based on above assay
  • Used glucose as the eluent from now on
HP and Modelling
  • Meet up with Washington iGEM team and came up with the idea to do a podcast
Nanobody surface display
  • Creation of fuGFP variants (fuGFPy, fuGFPb, and fuBFP) via PCR
  • Cloned variant plasmids into E. coli
  • Ran fluorescence plate to characterise the above variants
fuGFP-CBD
  • Ran SDS-PAGE and fluorescence plate of fuGFP-linker-CBDcipA using NT as the buffer and eluting 4 times using glucose
  • Sent SDS-PAGE of eluted bands for mass spectrometry
  • Ran SDS-PAGE and fluorescence readings of fuGFP-CBD variants (fuGFPb, BFP, fuGFPy) lysate and elutions
HP and Modelling
  • NSW iGEM team meetup on zoom
  • Modelling completed, and nanobody-GFP complexes are visualised in PyMOL
Nanobody surface display
  • Nanobody-GFP binding assay (see Protocols)
  • Sequenced original nanobodies, different fuGFP variants, and shuffled nanobodies
fuGFP-CBD
  • Repeated SDS-PAGE and fluorescence readings of fuGFP-CBD variants with one sample changed as it did not have an elution band on the gel
HP and Modelling