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Team:William and Mary/notebook
From 2014.igem.org
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- Set up stock solutions and prepared lab for a summer of awesome
- Attended school-mandated safety meetings and training
- Acquired samples of RCaMP and GCaMP from glycerol stocks from the lab of Dr. Margaret Saha
- Received ChR2 from Addgene and CatCh from the lab of Dr. Peter Hegemann, Humboldt-Universität in Berlin
- Transformed Aequorin (K548000) from Distribution Kit
- Began attempting Site Directed Mutagenesis (SDM) on ChR2's and RCaMP's internal PstI cutsites
- Brought up and transformed GFPs (E0040 and K895006) and Double Terminator (B0015) from Distribution Kit
- Attempted more SDMs of RCaMP
- Added BioBrick prefix and suffix to RCaMP with PCR
- Attempted to ligate RCaMP into pSB1C3
- Analyzed sequence data from parts worked with so far
- 3A assembly of Aequorin, GFP, YF1/FixJ to Double Terminator (DT)
- Performed SDM on CatCh that unfortunately failed
- 3A assembly of Bacterial Promoter-RBS (BP-RBS) from kit to Aequorin-DT, GFP-DT, and YF1/FixJ-DT
- Added BioBrick prefix and suffix to CatCh with PCR
- Ligated CDRE and Kozak sequence into pSB1A3
- Began amplifying BioBrick backbones
- Redesigned primers in an attempt to get ligations to work. This was a step in the right direction!
- Performed second SDM on CatCh. It looks like both PstI cutsites were removed
- PCR'd on prefix/suffix onto RCaMP, CatCh, and ChR1
- SDM on ChR2 to mutate out PstI cutsite. This did not appear to work
- 3A assembly of CatCh, RCaMP, and ChR1 with DT and ADH1 terminators, and subsequently BP-RBS to Aequorin-DT, GFP-DT, CatCh-DT, ChR1-DT, and RCaMP-DT
- Ligated CatCh, ChR1, and RCaMP into pSB1C3
- Performed PCR to add on prefix/suffix to ADH1 promoter
- Ordered a box of Fisher pens
- Analyzed sequence data of first set of 3A assemblies. Nothing was successful, but CatCh was confirmed!
- 3A assembly of ADH1 and Kozak sequence
- Attempted to perform SDMs on ChR1's and CatCh's first internal PstI cutsite. ChR1 appeared to be successful on a gel!
- Tested restriction enzymes to check to make sure they were not causing problems in ligations
- Created detailed records for our reference of sequencing data, inventories, and clone sheets
- Tried a gel purification on parts in preparation for ligation. Resulting nanodrop values were too low to be used
- Analyzed sequence data from 3A assemblies. Numerous important assemblies were successful!
- Ordered gBlocks gene fragments of important parts, including functional units of ChR1, CatCh, and RCaMP
- Began performing test ligations and assemblies from various parts from the distribution kit, in order to better understand why our ligations with our parts were failing. We varied digestion and ligation times and whether or not we purified parts
The College of William & Mary does not allow students to live on campus during the month of August. We worked on analyzing sequence data and preparing for the Jamboree from home.
- Attempted to ligate gBlocks into pSB1C3 and pSB1A2 (in order to be able to combine various genes into the same organism)
- Tried ligations again, this time using CIAP to prevent the backbone from ligating to itself and excluding the part - unsuccessful; CIAP is difficult to deactivate
- Ordered primers to amplify gBlocks
- Focused on RCaMP and ChR1 gBlocks
- Attempted to amplify gBlocks and ligate into pSB1C3 using various experimental conditions
- Continued trying different conditions for amplification and ligation of RCaMP and ChR1 gBlocks
- Tried using Shrimp Alkaline Phosphatase (rSAP) - unsuccessful
- Submitted ChR1 and CatCh in pSB1C3 to the Repository
- Worked on presentation and wiki
