Notebook: Week 8
Preparations | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16
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Aims of the Week
The Lab team are aiming to begin assembly of Ribosome Binding Site and Constitutive Promoter, which could then be used for a number of our modules. James and Leonard have also been working towards characterising salt tolerance, and aim to begin doing this by the end of the week. The protocol for this experiment has been decided, and so it is likely we will have some characterisation data by the end of the week. Rhiannon's aim was to finish setting up the wiki LabBook, and publish it on the wiki. The Rathenau Debate Deadline is approaching, and Erin plans to make the final touches before it is sent off. Carina also wants to confirm her plans for the architectural project and begin a diary documenting her designs.
Monday 30th July
Wet Lab. After the concern that the small size of the BBa_J23119 and BBa_B0034 insert means we cannot truly detect the presence of the correct insert (Ext 7.3), Rhiannon and Aurelija decided to investigate other ways of detecting it. This included setting up a 25bp ladder on 3.5% gel, but neither the 12bp insert (BBa_B0034) or the 35bp insert (BBa_J23119) could be detected. This may be due to the low concentration of these plasmids - and so we are considering using PCR to amplify them.
Meeting - wiki. Rhiannon met with many of the team members to discuss the design for the Lab Book page. Our aim was to make the work description sufficiently detailed that it could be easily reproduced by others in the future. At the same time, we risked making it too long to read. For this reason, we have a summary infographic for each class of experiments, which clearly illustrate the method and results. However, we have also contained all of the necessary information in a drop-down from this image. While the design will continue to evolve, we are currently content with it at present.
Tuesday 31st July
Wet Lab. Todays activities included PCR reactions of plasmid backbones, to amplify them before use, and analysis of their concentration by nanodrop. This proved somewhat disappointing. There was also miniprep of our Tetracycline Repressor (BBa_C0040) and the Gas Vesicle Cluster (BBa_I750016), but both proved to have very low [plasmid concentration. We also grew up some of our W3100 cells transformed with the Salt Tolerance BioBrick (BBa_K398108), as well as some untransformed W3100s, for characterisation of salt tolerance tomorrow.
Rathenau. Erin finished her write up of the Rathenau debate, and after proof reading from other members of the team sent it to an advisor for assessment.
Wednesday 1st August
Wet Lab:Today James began characterisation of the salt tolerance BioBrick. We used a simple protocol, involving 3 different salt concentrations for a transformed and untransformed line of W3100s. We also commenced 3A assembly and transformation of our Constitutive Promoter (J23119), the stress promoter (PcstA), the Ribosome Binding Site (BBa_B0034) and the plasmid backbone PSB1K3.
An analytical digest of our plasmid backbones proved disappointing, as so we began a repeat of the PCR. We also re-picked colonies of the BBa_C0040 and BBa_R0040 BioBricks, which have proven very difficult to transform and culture.
Modelling - meeting with Chris Barnes: Chris Barnes is a member of UCL staff who created the ACBsysBio software during his days in Imperial. We had already talked to Chris Barnes about how his software was going to be useful for our project. This meeting was to check how our initial model, written in MATLAB SimBiology, would be transported across into his software. Outcome: It appears the the model will work.
Human Practice -DIYbio Collaboration:This evening Yeping and Martina visited the London Hackspace again to give the 'biohackers'/citizen scientists a short introduction to synthetic biology and the iGEM competition which was requested last week during our safety session. We have set a facebook group for the collaboration so we can exchange resources and update progress on the collaboration.
Human Practice - Visit to Southbank:The aim was to inform and conduct interviews to see what people think of our project idea. Also a prototype of an island in plaster was on display allowing people to put flags on the island to name their own piece . This collaborative art display got many children interested in our project. People are interested in what we are doing in the lab and are concerned about the containment aspects with regards to deliberate release of GMOs into the environment.
Thursday 2nd August
Wetlab: James completed the protocol for the salt characterisation, which appears to have been successful. This will be repeated early next week. There was also a gel for the repeat of the PCR of plasmid backbones, which again proved unsuccessful - for reasons that are not clear to us.
Also given the problems we have had with transformation - we have set up another series on transformations, which will attempt to eliminate some of the causes of problems already experienced. We are also allowing only two members of the team to carry out the protocol from start to finish, to limit the number of possible problems. BioBricks that are being transformed are the Constitutive Promoter (BBa_J23119) and Ribosome Binding Site (BBa_B0034) due to their low concentration. Also included is a a spare Constitutive Promoter (BBa_J23100) and a spare Ribosome Binding Site (BBa_B0030)
Meeting - wiki. Rhiannon and Philipp met for several hours to finalise all important aspects of our lab book before publishing it to the wiki.
Friday 3rd August
Meeting - Speed debating Bethan met Steve Cross from the Public Engagement Department to get advice on advertising the event. Martina, Yeping and Philipp met
Dr Tarit Mukhopadhyay from the Biochemical Engineering to get feedback on the whole event.
Human Practice-Speed Debating Bethan, Martina, Philipp and Yeping discussed the final format for the upcoming speed debating event so it is both engaging and fruitful. They then spent the afternoon emailing around to get last minute advertising out there. Almost reached 80% sign-up on Eventbrite!
Saturday 4th August
Sunday 5th August
Meeting - Construction Aurelija and Bouran met to discuss the issues remaining in construction. This included discussion of which BioBricks we should reattempt transformation, and making she we have all the primers we need to progress.
Reflections
The team is still feeling restricted by the rate of the construction. We seem to be encountering numerous problems with transforming certain BioBricks (Expt 7.4 & 8.1), and we cannot confirm if our first attempt at assembly (Expt 8.4) has worked since the miniprep concentrations were too low and it did not show up on the gel. However, we have moved onto the characterisation stage for salt tolerance (Expt 8.3) which has been encouraging, as the experiments appear to demonstrate that our transformed cells are salt tolerant. We are also pleased to have produced the first complete draft of the Rathenau Debate, which has been sent off for assessment.
Success of the Week. Characterisation of Salt Tolerance
Fail of the Week. 3A Assembly
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