Contents
Week 0
Pitch ideas. Narrow down the ideas we liked- A BIOSENSOR FOR CAMPYLOBACTER. How do we do it? Research biology.
Weeks 1 and 2
Molecular biology boot camp- refreshing and learning all the techniques needed for our project. Finalising sub-projects. Further research. Health and Safety Training . Prep for Edinburgh meetup.
Week 3
Edinburgh meet up.
Biology: The biobricks needed were washed off the distribution plate and transformed. Created and ordered primers for the araC protein. Ordered WT mtle intergenic region and minimal promoter with B0032 and minimal promoter with B0034.
Engineering: Working on heater part of extraction step. Need 1% acetic acid and sample i.e. swab and mix the 2 alongside heat (100 degrees). Trying to make heater with a negative feedback loop. Small heater that can be worked with small liquids. Wrote code for temp controller and heating compartment. Temp monitor made and make heating element works.
Human Practices/ Outreach: Article sent to the campus newsletter. Talked to Chris French who gave info on new legislation for GMO biosensors.
Week 4
Biology: Lots of further transformations and minipreps of biobrick plasmids. Started digesting and ligating our parts together. Ordered PCR primers for araC and xylulose synthesis. Started the Interlab.
Engineering: Started making device which will process sample and give us signal. Made a heater and made a detector of light.
Human Practices/ Outreach: Skyped Deborah Scott; works with regulatory issues in synthetic biology. Gave us idea on what we could do with human practices; suggested reaching out to regulators for our project as we want to address either private audiences or private kitchens, farms etc. We need to know safety regulations around processing and packaging and testing chicken so we should reach out to them. Why is 70% contaminated? Could we go to a chicken farm or agricultural college? Asked agricultural college about making a biosensor. We should think about implications of Brexit.
Week 5
Biology: Continued ligating our parts together. Having issues with the constitutive promoter+ tetR ligation, is not transforming. Some low transformation efficiencies. Minipreps sent for sequencing. Started work on AND gate.
Engineering: Talked to Melanie completely changed the design and simplified to make it easier for everyday use. Kit Design: 3 Eppendorf tubes with different contents. You will swab the surface, dip it in tube #1, heat the sample, and then transfer between the other tubes. Then place it on a paper strap that will have our bacteria on it, a control etc, and will give colour. We made a product instructional leaflet.
Week 6
Biology: Continued plasmid construction. Started mutagenic PCR of araC, however getting low concentrations of product. Got tetR system to work- confirmed with sequencing. Decided to change from chromoprotein to GFP for quorum sensing. PCR for AND gate.
Engineering: Another change: all contained within one device, user is not in contact with acid/ alkaline etc. Two teams- bacteria on paper, the rest of the device. Make Xylulose labile, neutralise acid, mix with glucose, detection stage, device will do all these things, different chambers, swab will do in device and then break to release acid, pushed through chambers.
Human Practices/ Outreach: Paul Ellwood skype call: no new info, explained STIR protocol- integrate social sciences and science. How project will affect the world. (another skype call with STIR people, Eric Fisher). Arranged a skype call with ACT campaign. Science centre finalised. Explorathon application submitted. GIST article submitted. Podcast done with institute Collated attributions page. Started BREXIT chapter.
Weeks 7 and 8
Biology: Finished the Interlab. Started extra-credit experiments. Plasmids coming along well. Most final constructs transformed, miniprepped and sequenced. Some mutations. araC sub team decided to use the LacI regulated promotor, the strain will cause this promoter to be activated.
Engineering: Started making hydrogels to immobilise the E. coli using polyacrylamide. Printed paper with wax to enclose the gel. Tested the viability of cells in the hydrogel, then taking some and spreading them on a plate. Laser cutter tests, needed to redesign the plate as it was warping. Set out a plan for testing the tolerance of E. coli to tris acetate. Model of the channel that the liquid will flow through.
Human Practices/ Outreach: Had a phone call with acting with campylobacter campaign- very useful, all from a scientific background, seemed very interested in the project. Chlorine water chicken links to Brexit, might import from the states. UK is okay with it, however focus groups have said that it’s not good and people wouldn’t be happy with it. Can be linked to cancer.
Week 9
Biology: Most plasmids fully finished. Still some issues- araC mutagenesis PCR worked well but transformation failed, only two colonies but aiming for 160,000. Quorum sensing parts don’t work, ordered new parts, varying parts of intergenic region from E. coli. They naturally have 2 promoters going separate ways. Looking at full promoter, part of it was used by Tokyo team, they said it works but didn’t test it enough. So, ordered that part and another with another region. Another part been ordered with an even greater tract of DNA including area where LssR will bind. Ordered as 2 ultras and one with a primer to do PCR with. Second plasmid ready for promoters to be added once they arrive. Finished Interlab extra credit.
Engineering: Performed a test to see if gel could adhere to paper strips. Rueben planning to test for effects of salt concentration for neutralisation step of sensor and 3D modelling the fluid path.
Human Practices/ Outreach: spent two days at the Science Centre – kids and adults enjoyed the activity that we prepared for them. We had a chance to educated families about chicken preparation and cooking.
Week 10
Biology: Final plasmid constructions. Tried to optimise araC mutant library PCR, may need higher concentration for transformation. Tried TOPO TA cloning with little success. Quorum sensing Annealed the ultramers, added them to PSB1C3, confirmed. PCR to make the larger intergenic region, too big to make as an ultramer. First PCR failed, second done with varying temps, and that worked. Started testing the AND gate.
Engineering: Helped Edinburgh iGEM team with syringe pump system. Did calculations to make the fluid pump produce the right flow rate when entered into their system. Started to sporulate bacillus. Trying to re-do cell viability test, putting bacillus in a gel then introducing an Xgal solution, will turn blue if viable. Viability tests with Tris Acetate. Tested microfluidic plate, unfortunately “gunks” at the pot. Channels don't leak however which is good. Started designing a case for the device.
Week 11
Biology: Parts prepared for submission. Final transformations. More testing on AND gate. Started testing for Mtle and quorum sensing. AraC library still not ligating and transforming, tried ethanol precipitation of DNA and electroporation. Achieved a successful transformation with many colonies.
Week 12
Biology: Final testing of all our systems. Miniprepped araC mutant library, transformed, screened and tested. Submitted our biobrick parts to igem.
