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Contents 1 Team CRISPR 1.1 July 3 1.2 July 4th 1.3 July 5th 1.4 July 6th 1.5 July 9th 1.6 July 10th 1.7 July 10th 1.8 July 11th 1.9 July 12th 1.10 July 15th 1.11 July 17th 1.12 July 22nd 1.13 July 23rd 1.14 July 24th 1.15 July 25th 1.16 July 26th 1.17 July 29th 1.18 September 16th 1.19 September 18th 1.20 September 19th 1.21 September 20th 1.22 September 21st 1.23 September 22nd 1.24 September 23rd 1.25 September 24th

Team CRISPR

July 3

List of BioBricks that are/will be on the way:

Cas9:

• Cas9 - Joe and Anna
• pTet -
• const. promoter + Cas9 - Joe
• promoter + Cas9 + Leader + R + spacer + R (on Kan)
• L + R + spacer + R

Target:

• tracerRNA
• repeat + spacer + repeat -
• leader - Ray, Fisal, Dan

Decoy:

• PAM + spacer (on Amp)

July 4th

Experimenter: Cam Strachan

Aim: Phosphorylate and assemble T4, T7, and tracrRNA ordered oligos for decoy plasmids.

Results: Oligonucleotides were assembled using the assembly from annealed oligos protocol.

July 5th

Experimenter: Cam Strachan

Aim: Ligated annealed oligos into pSB1C3, cut with E and S, and transform into 10G heat competent cells. Cells were recovered in 300 µL and 25 µL was plated along with a no insert control.

Results: NA

July 6th

Experimenter: Cam Strachan

Aim: Select colonies for screening

Results: Approximately 100 colonies were present for both the T4, T7 and tracr RNA spacers with approximately 10 colonies on the background plate. Colony PCR with VF and VR2 confirmed bands at approximately 350bp. 3 colonies from both T4, T7 and tracrRNA plates were sent for Sanger sequencing through Genewiz.

July 9th

Experimenter: Cam Strachan

Aim: Phosphorylate, ligate and amplify oligos for T4 and T7 repeat-spacer arrays.

Results: The T4 and T7 spacers, repeat and the Repeat-BB Suffix oligonucleotides were phosphorylated with T4 PNK.

All oligonucleotides were mixed in equimolar amounts at a final concentration of 10 nM each, annealed and ligated and PCR amplified according to the repeat-assembly protocol.

July 10th

Experimenter: Anna Müller

Aim: PCR the cas9 gene from Streptococcus thermophilus cells.

Results: Unsuccessful no bands were seen on the agarose gel.

July 10th

Experimenter: Cam Strachan

Aim: Gel purify assembled sequences.

Results: The PCR product from yesterday was loaded into a 10% TBE-PAGE gel and run at 100V until the blue marker ran off. Bands corresponding to one (148 bp) and two (219 bp) were excised and purified.

July 11th

Experimenter: Cam Strachan

Aim: Finish purification, cut, ligate and transform repeat-spacer assemblies

Results: Gel slurries were filtered and ethanol precipitated and quantified. Products were cut with E+S, ligated into PSB1C3 and transformed.

July 12th

Experimenter: Cam Strachan

Aim: Finish purification, cut, ligate and transform repeat-spacer assemblies

Results: Gel slurries were filtered and ethanol precipitated and quantified. Products were cut with E+S, ligated into PSB1C3 and transformed.

July 15th

Experimenter: Cam Strachan

Aim: Screen/confirm insert length.

Results: 5 colonies from the single insert and 10 colonies from the double insert plate were screened via colony PCR for correct insert length. Three positive colonies from the single insert and 6 positive colonies from the double insert were inoculated for overnight cultures.

July 17th

Experimenter: Cam Strachan

Aim: Figure out why no T7 sequences were assembled.

Results: Sequencing results showed that the T4 spacer was correctly assembled into the repeat-spacer-arrays for single and double inserts. Unfortunately, no T7 spacers were incorporated in the assembly. After going over the sequence of the oligonucleotides with Mike, this is most likely because one of the T7 splint sequences was not correctly designed; we accidentally left the PAM site in it (which would have prevented ligation). The splint was redesigned and reordered; also designed and ordered a second set of different T4 and T7 spacers.

July 22nd

Experimenter: Cam Strachan

Aim: Restart assembly with fixed splint

Results: The repeat-spacer assembly was restarted with the fixed T7 splint and the new spacers and splints. New splints were phosphorylated with T4 PNK.

All oligonucleotides were mixed in equimolar amounts at a final concentration of 10 nM each, annealed and ligated and PCR amplified according to the repeat-assembly protocol.

July 23rd

Experimenter: Cam Strachan

Aim: Gel purify assembled sequences.

Results: The PCR product from yesterday was loaded into a 10% TBE-PAGE gel and run at 100 V until the bromophenol blue marker ran off. Bands corresponding to one (148 bp) and two (219 bp) were excised and purified.

July 24th

Experimenter: Cam Strachan

Aim: Cut, ligate and transform repeat-spacer assemblies

Results: Gel purification products were quantified with the Qubit. Single inserts for T7 and the new T4 and T7, and double inserts for T4+T7 (original set) were cut with E+S, ligated into PSB1C3 and transformed.

July 25th

Experimenter: Cam Strachan

Aim: Screen/confirm insert length.

Results: 5 colonies from each of the single insert and 10 colonies from the double insert plates were screened via colony PCR for correct insert length. Two positive colonies from each of the single insert and 6 positive colonies from each of the double inserts were inoculated for overnight cultures.

July 26th

Experimenter: Cam Strachan

Aim: Miniprep and prepare for sequencing

Results: Miniprepped overnight cultures from yesterday and send to sequence confirm.

July 29th

Experimenter: Cam Strachan

Aim: Analyze sequencing results

Results: Based on the sequencing results, the single insert T7, new T4 and T7 and the double T4 and T7 inserts were correctly assembled. The constructs were given to Joe to add promoters and a terminator via standard assembly.

September 16th

Experimenter: Cam Strachan

Aim: Set up T4 phage growth curves.

Results: Added varying concentrations of phage based on Dan’s plaque assays to different concentrations of 10G cells inoculums. Plate all starting concentrations to get CFUs for inoculums. Doing this all in a 96 well plate on the TECAN in the Kiefer lab.

September 18th

Experimenter: Cam Strachan

Aim: More T4 growth curves.

Results: Ran more inoculums. Set up the machine to run longer because we weren’t reaching lag for a bunch of isolates. Also shook the plate more to get more oxygen in there.

September 19th

Experimenter: Cam Strachan

Aim: Ran growth curves again start the combinatorial library stuff (see protocol).

Results: More inoculums wooooo. Digest gel looked good. Equilibrated the gel purified product concentrations.

September 20th

Experimenter: Cam Strachan

Aim: Pick colonies into a library.

Results: Picked two 384 well plates worth of colonies and added 10 µg/ml of arabinose. I then added 10³ phage. Will screen for growth tomorrow.

September 21st

Experimenter: Cam Strachan

Aim: Read OD of combinatorial library.

Results: There seems to be some hits, set them up under the same concentrations with 4 different arabinose concentrations. See if survival is arabinose dependant.

September 22nd

Experimenter: Cam Strachan

Aim: Read OD induced survivors

Results: There looks like there is a trend of survial at intermediate arabinose concentrations.

Experimenter: Anna Müller

Aim: Digest T4, T7 spacer sequence with PAM site(decoy construct) with EcoRI and SpeI to later on ligate into plasmid with ampicillin resistance.

Results: will follow.

Aim: Digest PsBIA3 plasmid with ampicillin resistance with EcoRI and SpeI to construct the decoy plasmides.

Results: will follow.

September 23rd

Experimenter: Cam Strachan

Aim: Picked out hits from screen into a 96 well plate. Did growth with 10^3 phage. Michael also helped me do 45 colony PCRs, they all have 5kb bands (except for a couple) suggesting that cas9 is in the contruct.

Results: Ran another TECAN run overnight.

September 24th

Experimenter: Cam Strachan

Aim: Analyze 5 tones of growth data.

Results: Looks like there is a partition between survivors an diers. Summarized all this data for joel to see if it fits the models.