"
Team:NRP-UEA-Norwich/Week6
From 2012.igem.org
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 at the Forum. 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 very excited about the UK meetup, despite the inhumanely early start. In our minds it 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 and 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 morale is on an all time high. Preparation for validation of our suspected BioBricks was started with researching sequencing companies and performing mini preps on the most promising colonies.
The Growth Study was repeated but was run for 9 hours instead of 6 hours. A similar procedure to last week was followed. LB broth media was inoculated with E. coli either transformed with PyeaR +GFP or not. To the media of these samples, a varying amount of potassium nitrate (0mM, 5mM and 10mM) was added. Both absorbance and wavescan readings were taken. Measurements were taken once every hour and in between placed in a 37 °C incubator. Instead of plating constantly, we changed our strategy to last week. We decided to initially plate alpha cells growing in media containing different concentrations of potassium nitrate. Using a culture of alpha cells we diluted to various concentrations, we took absorbance readings and plated them. Using these, we could then draw up a graph and calculate a calibration curve to 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 were in lag phase throughout the study. Their numbers did not reach a high enough level for readings to be taken without a large relative inaccuracy from the spectophotometer. The study will be repeated after tweaking 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 and repeated the process of a time period of 5 hours. Looking at the results, the readings were >2.5 and hence the actual absorbance was beyond the capacity of the spectophotometer. To redo this study, a starting concentration of above 0.2 would be better. To do this, rather than inoculating directly from plates, growing the cultures overnight and peletting then, then resuspending them in new culture would be easier and give better results. Alike inoculating from plates, the cells would be in stationary phase or be dormant, however, they would return to growth phase much faster. The plates inoculated from had been stored in the fridge and therefore, the cells were probably returning to growth phase in a longer period of time.
LB media and Agar media was made in vast quantities and autoclaved, to be prepared for further ligations later this week.
To produce more MB and BM 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 for filling 5.5ml inoculation jars.
Forum preparations - plates with "BioBrick" written one letter per plate (using PyeaR + GFP cells and potassium nitrate in the media to induce fluorescent activity) were prepared. 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 MB and BM that was similar to the protocol developed with PyeaR + GFP cells. However, they started on much higher concentrations of MB and BM 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 + GFP to MB and BM. For further information, the results can be found here.
Forum preparations - cross words, amino acid decoding sheets, comprehension sheets and word searchers were produced to offer activities to a large range of age groups. posters containing information about synthetic biology and iGEM were finished and printed. A summary of the day can be found on the Human outreach page.
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. Later on in the day, we rehearsed in front of advisors and the team.
Forum preparations - 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. Check out the Human outreach page 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 and watch videos of the life stream as well as pictures on the Human outreach page.
