Team:UCL/Notebook
Notebook
March & April
First team meeting. Members introduction and discussion about each members strength, potential contribution to the project and iGEM deadlines.Brainstorming some ideas for our project. We decided to pool DNA origami, to bioremediation to drug delivery vehicles and vote on them.
May
Week 3
Project choice: We decided to use encapsulins for drug delivery - we also planned to genetically fuse DARPins on their surface to make the encapsulins targeted drug delivery vehicles. Discussion about iGEM safety regulations and allocation of Safety Officer (Vlad)
Week 5Production of detailed project breakdown. Identification of elements required for a successful project and brainstorming, rough role allocation, decided on overall lab work objectives Supervisors meeting: disseminated all the various steps for the lab work and guidance about our human practices.
June
Week 7
Meeting with Departmental Safety officer Brian O’ Sullivan, modelling brainstorming, detailed discussion of cloning and troubleshooting on potential problems with our project with determination of objectives we wanted to achieve throughout the timelines up to the Jamboree, discussion of consumables and equipment required. London SynBio Network meeting - initial meeting with London’s other iGEM teams (Westminster and Kings) and insightful talks from current synthetic biology researchers.
Week 8Meeting with Green UCL’s representative Martin Farley - insight on sustainable lab practices. Discussion on how lab protocols that would contribute greatly towards resolving climate change. For instance, he told us that it would be more sustainable for researchers to recycle more at work than at home! Meeting with social media organiser for the biosciences department - Kim Morgan. Extensive discussion about cloning and purification of produced biobricks along with potential logos and team names.
Week 9Discussion on the content of the project inspiration. Decision on main vision, problems and shaping our project aim. Concluded that our project would involve developing a modular platform based on encapsulins/DARPin construct that has many potential applications that are not limited to the realms of drug delivery. Discussion for potential drug payload to induce apoptosis of cancer cells. Completion of safety form and further discussion on the project inspiration and cloning strategy. Weekly meeting!
Week 10Cell culture induction with Dr. Rana. Cloning workshop: help on construct design to keep our platform as modular and functional as possible. Meeting with Elpida Makrygianni to gain an insight into further public engagement that we could do. Made us aware of the importance of considering an appropriate audience and the variety of activities that we could engage with. Modelling meeting with Prof. Chris Barnes. Brainstorming on collaboration ideas, sustainable practices ideas, and preparation for our first public engagement event. Participated on Sustainable Development UN Goals challenge from team Costa Rica. Weekly meeting: Discussion on how to target medal criteria with Dr. Darren Nesbeth.
July
Week 11
First Day in the Lab! Meeting to discuss on launching our sustainability challenge in line with London’s Climate Action week! Meeting with the Chihuahua team from MexicoUploaded our sustainability collaboration link onto our wiki! Lab work: Transformation of DH5α E. coli strain with pSB1C3 for future cloning and BBa_K2842690 for plasmid amplification.Transformation of BL21* (DE3) E. coli strain with BBa_2842690 for scale-up studies for Bronze medal characterisation.
Week 12Took a deeper look into judging criteria by meticulously mapping the judging handbook to the various parts of our project. Drew up a paper prototype of our wiki - Troubleshooting on navigation bars for wiki with the help US AFRL and the Chihuahua team. Meeting with Gerald McBrearty from the EEE department to borrow electrical circuits for our public engagement workshop. Attended a talk by Jake Beal and discussions on standardisation for GFP and other fluorescent proteins. Came up with possible measurements collaboration idea to create a new system of calibration curve. Lab work: 37 oC 50 mL shake flask cultures for Intein monomer 2, BBa_2842690 , expression and solubility studies. Growth curves for BL21*(DE3) transformed with BBa_2842690, BBa_2842690's GFP reporter fluorescence measurements via plate reader for protein quantification. Optimisation of growth protocol for increase of BBa_2842690 expression and solubility. Transformation and blue-white screening for BBa_K3111101 expressing colonies and miniprep for plasmid amplification.
Contacted representative at UCL Research Ethics Committee and Breast Cancer Now to discuss potential Human Practices ideas. Conducted our first interactive session, Building with Biology, using electrical circuits. Met with Martin Farley to discuss on the progress of the Emerald Challenge along with feedback on potential improvements and expansion of our sustainability challenge as an integral part of the iGEM competition. Advice on how to initiate a case study to potentially influence iGEM to take action on making sustainability a criteria for ALL iGEM teams for the future. Skype call with Lydia Morrisson from NEB podcasts. Lab work: BBa_K3111101 50 mL shake flask expression and growth curve creation BBa_2842690 no induction 37 o C, induction 37 o C and induction 18 oC expression studies on solubility and quantification through fluorescence microplate readings and Western Blots IDT gBlock Delivery Troubleshooting restriction digestion and gel extraction.
Met-up to discuss particulars of the presentation for the London SynBio Showcase on the 23rd of July. Practised the presentation before Martina - our supervisor! Presentation at the London SynBio Showcase! An excellent opportunity to gain feedback on our project and to network with other researchers in SynBio. Meeting with Prof Oscar Della Pasqua, who is an expert in clinical pharmaceuticals. Meeting with KCL iGEM team for collaborative discussions. Meeting with Prof Steve Brocchini from UCL Pharmacology department. Lab work: Cell sonication, BBa_K3111101 FLAG-tag column purification and SDS PAGE analysis Transmission Electron Microscopy of M. xanthus encapsulins. PCR of IDT gBlocks and troubleshooting for unspecific bands. Gel extraction of correct size DNA fragment.
Finalised presentation for Newcastle iGEM meet-up. Meeting with UCL Research Ethics Committee and Breast Cancer Now as part of our Human Practices efforts. Another day spent collaborating and listening to interesting presentations by other UK iGEM teams at the Newcastle iGEM UK meetup. Newcastle iGEM meet-up continues. We gave our presentation today and continued our discussions and collaborations. Lab work: Delivery of Twist gBLocks and PCR amplification and cleanup Further PCR troubleshooting on IDT gBlocks Gel extraction of correctly sized DNA bands Cell-Free Protein Synthesis of BBa_2842690 and fluorescence spectrophotometric analysis for protein solubility
August
Week 16
Meeting with Dr Yin Wu - a clinician who gave us an insight into how our product might be received by clinicians and provided key feedback that we incorporated into our project design. Meeting with Elpida to evaluate our progress with public engagement, and to discuss future outreach events Weekly meeting Lab work: M. xanthus encapsulin physicochemical properties using Dynamic Light Scattering Transformation, expression of BBa_K3111201 and BBa_K3111202 and purification Ligations of PCR amplified gene fragments (Twist,IDT) for transformations Gel extraction yields troubleshooting
Meeting with Dr Michael O’Neill - industry expert who gave us specific feedback on testing the design of our drug delivery platform. Weekly meeting with detailed discussion around the medal criteria that we have completed until now and further plans. Lab work: SDS PAGE and Western Blot analysis of purified BBa_K3111201 and BBa_K3111202. Incubation of purified BBa_K3111201 and BBa_K3111202 with SKBR3 HER2 overexpressing cells to determine optimal protocol for incubation and binding Wester Blot of CFPS expressed BBa_2842690 . M. xanthus encapsulin stability studies using Dynamic Light Scattering Repeat Ligation of additional biobricks into pSB1C3 and transform into DH5α
Meeting with Sara Brouwer from the Institute of Making, who gave us many ideas on potential outreach activities and gave us feedback on our previous public engagement event (Virtual Schools). Meeting with Prof Paula Lorgelly (health economics) who gave us valuable insight into the healthcare system and how our project might be influenced by specific economic policies in the future. Meeting with Westminster iGEM for Emerald challenge collaboration! Meeting with Dr Ahad Rahim who gave us general advice on our project. Weekly meeting! Lab work: Amplification of BBa_K3111104, BBa_K3111501, BBa_K3111102, BBa_K3111103, BBa_K2686001, BBa_K2686002 plasmids and test digest to identify colonies with full length plasmid. Transformation of the correct plasmids into BL21*(DE3) and sequencing. Incubation of BBa_K3111201 and BBa_K3111202 with SKBR3 cells under optimised protocol, to compare binding of the two constructs on HER2 receptors at different temperatures and applied concentration.
Meeting with Westminster. Elpida Makrygianni spoke to the Royal Academy of Engineering who were willing to share a stall with us at New Scientist Live. Call with Dr Nihal Sinha who gave us important stakeholder feedback on our project design, drawing on his experience as a clinician Interview with Professor John Ward, Dr Stefanie Frank, Noelle Colant for NEB podcast Weekly meeting! Lab work: 50 mL shake flask culture for expression of BBa_K3111104, BBa_K3111501, BBa_K3111102, BBa_K3111103, BBa_K2686001, BBa_K2686002 Purification of BBa_K3111102, BBa_K3111103 and SDS PAGE analysis for solubility Heat purification of BBa_K2686001, BBa_K2686002 and SDS PAGE analysis for solubility Identification of compatible plasmid for cotransformation with pSB1C3. This was done to compare loading capacity of T. maritima encapsulin by direct downstream fusion of the cargo or expression of it on different plasmid.
September
Week 20
Obtained Ethics approval from UCL REC - Breast Cancer Now survey development commences. Collected samples for our exhibition at New Scientist Live from Aurelie Fontan, Post Carbon Lab and BioHm. Interview with Prof Qasim Rafiq for NEB Podcast. Final recording with Lydia Morisson for NEB podcast. Meeting with representatives from the Royal Academy of Engineering to discuss exhibition at New Scientist Live. Weekly meeting! UCL Open day - aka the launch of our exhibition on Synthetic biology and the Senses. Lab work: 50 mL shake flask culture 18 and 37 oC for BBa_K3111102, BBa_K2686001 and BBa_K2686002 to optimise solubility and SDS PAGE for comparisons Dynamic Light Scattering to observe the change in size pattern of expressed T.maritima encapsulins produced from BBa_K3111102, BBa_K3111103, BBa_K2686001 and BBa_K2686002. Ligation of BBa_K3111103 and BBa_K3111501 to incorporate Dr. Steve Brocchine advice on encapsulin-DARPin929 loading. Amplification of pBAD30 plasmid for cargo loading studies and BBa_K3111104 for expression and use in mammalian cell culture studies
Emerald Challenge Collaboration with Oxford iGEM Survey released to the Panel by Breast Cancer Now. Weekly meeting! Lab work: Cloning of BBa_K3111302 (miniSOG), BBa_K3111301 (sfGFP) following two cloning strategies: 1) cloning in pBAD30 for cotransformation with pSB1C3 plasmid containing BBa_K3111501/BBa_K3111103, 2) Direct C terminal fusion downstream of BBa_K3111501 coding sequence. This creates BBa_K3111032 and BBa_K3111031. Transformation and test digest to select correctly sized plasmids for BL21* (DE3) E. coli transformation CFPS studies of BBa_K3111104 for protein quantification and potential input for in vitro production dynamic modelling 50 mL shake flask culture of BBa_K3111501 for Encapsulin with surface displayed DARPins expression, purification, and SDS PAGE analysis for observation of solubility Transmission Electron Microscopy of BBa_K3111102, BBa_K3111103 and BBa_K3111501 to observe assembly
Our delivery of Microsilk arrived (for our exhibition on Synthetic biology and the senses) - thank you Bolt Threads! Important (aka scary!) iGEM meeting! UCL iGEM goes to a fashion show! We spoke with some designers who were integrating synthetic biology into fashion to borrow some samples for our exhibition at New Scientist Live. Weekly meeting! Lab work: Purification and SDS PAGE and BBa_K3111104 and BBa_K3111031. Generation of pSB1C3 BL21*(DE3) 50 mL shake flask growth curve to input data into in vivo encapsulin production modelling. Cargo loading studies by expression studies of BL21* (DE3) strains. This was to confirm difference in loading capacities between: pBAD30_sfGFP and pSB1C3 containing BBa_K3111103, BBa_K3111402,BBa_K3111502 transformed cells,through expression, purification and fluorescent spectrophotometric analysis. Cloning for creation of BBa_K3111503, purification and loading capacity studies. Transformation and expression studies of genetically modified BBa_K2686001 and BBa_K2686002.
Week 23Last week in the lab :( Mammalian cell culture studies using SK-BR-3 HER2 overexpressing cells and Mesenchymal Stem cells (MSCs) as control. Involved studies to investigate the rate of internalisation and control studies to investigate the specificity of the targeting peptide of our platform i.e. DARPin929 using flow cytometry. Native PAGE used to observe cargo loading of our encapsulins. Expression and Cargo Loading studies of BBa_K3111402, BBa_K3111502 and BBa_K3111503 using fluorescent spectrophotometric analysis and SDS PAGE. Mammalian cell culture to study cytotoxic induction of our drug delivery platform (BBa_K3111502) and assess loss of cell viability of SKBR3 and MSCs as a control.
October
Week 24
Presentation at London SynBio Network at Imperial College London. Weekly iGEM meeting Teleconference with Dr Christopher van der Walle Survey results collected from Breast Cancer Now
Week 25Weekly iGEM meeting! Our exhibition on Synthetic Biology and the Senses was set-up at New Scientist Live (10-13 October)! Engaged with younger and older audiences by presenting the principles of Synthetic Biology, our summer project and talked about iGEM!!!