Team:Linkoping Sweden/Notebook
Notebook
Week 10-13
08.03 - 04.04Discussed possible project ideas. Had brainstorming sessions, did a lot of research and finally decided to work with desalination of seawater using cyanobacteria mainly inspired by the Shanghai 2015 team and an article published in Plant Physiology by J. Amezaga et al. titled Biodesalination: a case study for applications of photosynthetic bacteria in water treatment.
Week 15-18
12.04 - 09.05More in-depth research regarding our project and SWOT analysis. Decided on more aspects of the project such as possible usage of fast-growing bacteria to validate the parts and ways of removing the bacteria from the desalinated water.
Week 19-21
10.05 - 30.05Had a tutorial on how to use Benchling and construct BioBricks. Started preparing protocols for the lab work and researched possible ways to improve an existing BioBrick. The logo of the project was finished.
Week 22-24
31.05 - 20.06Lab work preparation. Continued designing BioBricks and working on protocols. Had the first meetings with experts in the field. The first week of the lab was mostly focused on learning the methods and preparing reagents. Decided to take part in the postcard project organised by iGEM Düsseldorf.
Week 25
21.06 - 27.06Received the cyanobacteria Synechocystis sp. PCC 6803 and prepared knock-in plasmids pEERM1 and pEERM4. Released the first episode of the podcast. Decided on a plan for the mathematical modelling part of the project.
Week 26
28.06 - 04.07Prepared BG-11 media for Synechocystis sp. PCC 6803, restreaked them and prepared large-scale liquid cultures. Prepared our two other bacteria, E. coli and V. natriegens, for future transformations (competent cells). Continued contacting experts that could help in the improvement of different parts of the project and worked on our Integrated Human Practices.
Week 27
05.07 - 11.07Restreaked more wild type Synechocystis sp. PCC 6803 as well as the S-layer knockout from Synechocystis sp. PCC 6803. Transformation of our improved part started and several BioBricks studied for our contribution, chromoproteins, were transformed into E. coli. Liquid cultures of V. natriegens and E. coli were made with and without a beta-carotene 15,15'-dioxygenase-containing plasmid for control purposes. Started planning the Nordic iGEM Conference together with the Chalmers-Gothenburg team.
Week 28
12.07 - 18.07Visited Gotland to learn more about the water shortage on the island and talked to multiple potential users, farmers. Also collected seawater samples for our experiments. After returning, performed mutagenesis to correct mutations in eforRed improved DNA construct and then transformed it into E. coli XL1 competent cells. Started testing the transformation protocol on Synechocystis sp. PCC 6803 with pEERM1 and pEERM4 plasmids. Began our first experiments on ideal growth conditions of Synechocystis sp. PCC 6803.
Week 29
19.07 - 25.07Ran an agarose gel on the eforRed improved DNA construct to confirm that the mutagenesis was successful. Purified the pEERM1 and pEERM4 plasmids that were amplified in XL1. Continued experimenting with the Synechocystis sp. PCC 6803 transformation. Set up more growth experiments with different seawater samples and different conditions (different pH, salinity levels etc). Continued working on developing the modelling part of the project and on the podcast.
Week 30
26.07 - 01.08Transformed CBD-containing plasmids into XL1 for amplification several times due to unsuccessful attempts. Amplified linear constructs from TwistBioscience with PCR. Prepared plasmids with CBD, eforRed improved (both correct and mutated versions – to confirm that mutagenesis was successful). Sent several BioBricks from the distribution kit (that we had transformed) for sequencing. Had a meeting with SUNY Oneonta and TU Darmstadt teams. Hosted the Nordic iGEM Conference together with the Chalmers-Gothenburg iGEM team.
Week 31
02.08 - 08.08Continued working on CBD-containing plasmids with mutations because the previous mutagenesis was unsuccessful. Decided to order linear DNA fragments instead of performing mutagenesis to save time. Cloned MscL fragments into pEERM1 and pEERM4 plasmids, transformed into XL1 and verified the size of the obtained plasmids by gel electrophoresis. Transformed halorhodopsin pUCIDT plasmids into XL1. Started an experiment to monitor the growth of Synechocystis sp. PCC 6803 in autoclaved and non-autoclaved brackish and seawater using BG-11 1X as a control to obtain data for the modelling group. Continued with the transformation attempts in Synechocystis sp. PCC 6803.
Week 32
09.08 - 15.08Did colony screening of MscL in pEERM1 and pEERM4, beta-carotene in pEERM1 and pEERM4, and linker-GFP in pEERM1 and pEERM4 in XL1. Acquired data from chromoprotein characterization. Attempted to ligate linear CBD fragments into pEERM1 and pEERM4 and transform them into XL1. Continued with daily growth measurements of Synechocystis sp. PCC 6803 in multiple types of seawater and BG-11 media. Acknowledged that the bacteria in non-autoclaved water did not survive long. Took microscopic images of seawater samples with no added Synechocystis sp. PCC 6803 to find out what was killing the strain during the growth experiment. Established that the transformation attempts in Synechocystis sp. PCC 6803 appeared to be unsuccessful.
Week 33
16.08 - 22.08Continued working on the colony screens that were started last week, and then plasmid prepped and made glycerol stocks of the colony screening products. Attempted to transform pEERM1 and pEERM4 into Synechocystis sp. PCC 6803 following an alternate protocol. Ligated several parts with linker-GFP, like MscL in pEERM4. Continued with daily growth measurements of Synechocystis sp. PCC 6803 in multiple types of seawater and BG-11 media. Held a presentation during a Phototroph Meetup - meetings between various iGEM teams working with phototrophs.
Week 34
23.08 - 29.08Decided to treat cleaved plasmids with calf intestinal phosphatase before ligation in order to avoid re-ligation of the plasmid without the insert. Switched to using pSB1C3 and pUC19 as cloning vectors after discovering a design flaw using pEERM1 and pEERM4. Transformed halorhodopsin in pUCIDT plasmid and MscL-Linker-GFP in pEERM4 into V. natriegens. Continued with daily growth measurements of Synechocystis sp. PCC 6803 in multiple types of seawater and BG-11 media. Full-time lab work ended due to everyone going back to their studies.
Week 35
30.08 - 05.09Started the transformation of MscL in pEERM1 and pEERM4 into Synechocystis sp. PCC 6803 using the alternate protocol and continued working on cleaving and ligating our linear DNA fragments into pUC19. Continued with daily growth measurements of Synechocystis sp. PCC 6803 in multiple types of seawater and BG-11 media.
Week 36
06.09 - 12.09Ended with daily growth measurements of Synechocystis sp. PCC 6803 in multiple types of seawater and BG-11 media. Visited high schools as part of our Education and Communication.
Week 37
13.09 - 19.09Continued working with our linear DNA fragments, attempting to cleave and ligate them into pUC19 plasmids. Attempted to transform our constructs into E. coli.
Week 38
20.09 - 26.09Sent plasmids for sequencing to check if they contained the correct inserts. The transformation of MscL in pEERM1 and pEERM4 into Synechocystis sp. PCC 6803 turned out to be unsuccessful.
Week 39
27.09 - 03.10Continued working with our linear DNA fragments, attempting to cleave and ligate them into pUC19 plasmids. Attempted to transform the cloned constructs into E. coli. and V. natriegens. Studied the seawater samples with Synechocystis sp. PCC 6803 from previous growth experiments under the microscope.
Week 40
04.10 - 10.10Focused on perfecting and characterizing our improved part. Continued working on developing the wiki page and the presentation video.