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Team:NRP-UEA-Norwich/Week6
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Revision as of 16:33, 24 September 2012 by LukasHarnisch (Talk | contribs)
Welcome to the NRP UEA iGEM 2012 Wiki Lab Book
Please choose the relevant link to access our diary of that week!
Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments
Contents |
Much of this week was focused on both the UK team meetup and the human outreach event that we were putting on that Sunday. We started the week on a high with the production of our first BioBricks. The team split up into groups to concentrate on different aspects of characterisation and generation of other BioBricks. By Friday, everyone was super hyped about the UK meetup, despite the inhumanely early start. The UK meetup was a huge success. The forum was no different, we met and interacted with people of all ages, introducing them to synthetic biology and our project.
Forum Preparation were starting off with the design of posters, flyers, ordering consumables.
The liason with GenScript about construct synthesis continued .
We have BioBricks! The plates containing the ligations between B-M and M-B into pSB1C3 showed growth. We were very excited and the lab moral is on an all time high. Preparation for validation of our suspected BioBricks was started with researching sequencing companies and mini preps.
The Growth Study was repeated but was run for 9 hours instead of 6 hours. A similar procedure was followed. Into 15ml of LB broth media, an inoculation of E.coli colony cells with or without transformation with PyeaR was added. Into these tubes, a varying amount potassium nitrate (0mM, 5mM and 10mM). Both absorbance and wavescan readings were taken. Readings were taken once every hour and between they were placed in a 37 degree celsius incubator. Instead of plating constantly, we changed our strategy. We decided to plate alpha cells initially at different concentrations within the media. Using a culture of alpha cells we diluted it at various amounts and took the absorbance readings and plated them. Using these we could then draw up a graph and work out the number of cells relative to the absorbance reading. However, this did not occur as smoothly as we had hoped. Furthermore, as the study only lasted for 9 hours, and the solutions inoculated from colonies and into 15ml of solution which was too dilute, the study did not produce the results we had hoped for. The cells we in lag phase throughout the study. Their numbers did not reach a high enough level within the culture for readings to be taken without a large relative inaccuracy from the spectophotometer. The study will be repeated after tweeking of the protocol. The samples were retained in an incubator to take final readings the next day to see whether they reached a high enough concentration.
The PyeaR samples that were retained from yesterdays growth study were used to take final readings. We pipetted 1ml into cuvettes and set the spectophotometers to take readings every minute for an hour and repeated the process of taking readings for 5 hours. Looking at the results, we realised that during the night, the cells had reached growth exponential stage and had gone into stationary phase.
LB media and Agar media was made in vast quantities and autoclaved to be prepared for further ligations later this week.
To produce more M-B and B-M BioBrick DNA (already ligated into pSB1C3 plasmids), more colonies were inoculated into LB media. We plan to maintain a running culture of this, whereby we inoculate this into fresh culture daily.
Nanodropping of the ligated BioBricks showed the following concentrations:
BM1: 171.3 ng/µl BM2: 149.4ng/µl BM3: 58ng/µl BM4: 105.2ng/µl MB1: 119.9ng/µl MB2: 108.9ng/µl MB3: 98.9ng/µl MB4: 102.4ng/µl
We will continue using these repeats of both BioBricks for further ligations and sequencing to have a fall back in case PCR and ligation has introduced mutations in some of the genes.
The preparation work for the forum event was continuing with finalising the posters and Joy went to the forum to draw a plan of where everything would go such as the 'design your own biscuity monster' activity station.
Finalising the orders of the synthetic genes and clearing the orders with finance.
Khadija prepared extra agar for autoclaving and Rachel made extra LB media and pippetted 5.5ml into jars for autoclaving.
Forum preparation - plates with Biobrick written with one letter per plate (using pYEAR + GFP cells and potassium nitrate in the media to induce fluorescent activity). The potassium nitrate was directly added to the agar media. This was simply done by using one end of a glass spreader that we kept sterilising between plating and wrote on the plates directly. We printed large letters and placed the letter beneath the plates to aid with the writing. As most of the letters are mirrored, this did not effect the writing, only the letter R needed to be written freehand.
Lukas and Pascoe carried out a growth study on M-B and B-M that was similar to the protocol developed with PyeaR + GFP cells. However, they started on much higher concentrations of M-B and B-M cells in the cuvettes. Looking at the results of these we found no noticeable difference between the growth pattern of Bioline alpha cells and our BioBricks. However, as the starting concentrations/absorbances were very varied, we decided to repeat this when repeating PyeaR and at the same time compare PyeaR to M-B and B-M.
Forum preparation - cross words, amino acid decoding sheets, comprehension sheets and word searchers were produced to offer activities to a large range of age groups. Links to these will be uploaded on to wiki.
Forum preparation - posters containing information about synthetic biology and iGEM were finished and printed.
The project poster for the UK meetup was designed by Rebecca who used the helpful outline of the Forum poster made by Khadija.
Our presentation for the UK meet up was written and prepared and later on in the day rehearsed in front of the other iGEMers and our advisors.
Forum preparation - plates used for the forum science day had their fluorescence intensified by adding more potassium nitrate to the cells on the plate. Using an ioculation loop, the cells which had expanded beyond a clean outline of each letter was scooped away and inoculated into media. Using this media, with different colonies, a spare set of plates were made in much the same way as those created the day before. However, we double the concentration of potassium nitate from 40 to 80mM.
UK Team Meet-Up!
The team took the day off lab in order to host and enjoy a great UK iGEM team meet up at the google campus, London (Cheak out human practices tab for more information about this day).
The team left for the forum bright and early to set up and had a great day. Read about the forum from our human practice page.
