iGEM 2013 — Team Toronto

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# Team:Toronto/Notebook

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* [MODELING](https://2013.igem.org/Team%3AToronto/Modeling "Modeling")
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* [SAFETY](https://2013.igem.org/Team%3AToronto/Safety "Safety")

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###### **NOTEBOOK** **05/26** Prepared stock solution of LB and autoclaved empty bottles. **05/27** Prepared antibiotic aliquots and practiced pouring a gel. **05/28** Practiced dilution plating and glass bead plating. Worked collaboratively on a miniprep and used Nanodrop to evaluate the DNA purity. Met with Michael to talk about lambda red recombination. He is trying to make his own deletion strains with a pKD46 plasmid, recombinases, and PCR. Researched plasmid map of pBR322 to determine key characteristics like antibiotic resistance, origin of replication etc. Tried CV, Adhesion, CR and CF on 3 dilutions of overnight culture, grown for 48h. HindIII/NdeI digest of pMAL followed by gel electrophoresis and gel extraction of the large (5444bp) fragment for use in in-fusion. **07/24** **07/25** **07/26** Transformed MG1655 with pMAL glycerol stock via electroporation. Plated on LB agar with ampicillin. **07/27** **07/28** The solutions for the stimuli were all prepared: indole, ethanol, sucrose, salt and the buffered LB. They were autoclaved separately to prevent precipitation and mixed together. This was done a couple of times and perfected based on the solubility and other properties of the solutions. Set up plate plans for the strains. **07/29** **07/30** 2 WT overnight cultures in phosphate buffered LB, 50% EtOH solution was prepared using phosphate buffered LB as a solvent. 2 M sucrose, 1 M indole, and 1 M NaCl was also prepared in a similar way but the indole solution failed as solubility for this substance is only 0.19 g/100 mL. A graph was created comparing weight measurements of standard weights on the fine balance and the regular balance. It was discovered that both were reasonably accurate. Made glycerol stocks of MG1655+pMAL. Conducted miniprep of pMAL. Conducted nanodrop of miniprepped pMAL. HindIII/NdeI digest of pMAL. **07/31** Gel electrophoresis of pMAL digestion and gel extraction of large fragment. Nanodrop of large fragment (poor yield). Developed a phases plan for the assays. 1. Validate the machine: does it read concentration or volume? 2. Validate working concentrations of dyes 3. Validate petri-dish based assays with dilutions 4. Validate assays themselves. **08/01** Constant amount, not concentration, matters for absorbance. See CV data on July 31. Another digestion of pMAL, along with extraction and nanodrop. Poor yield once again. Started another overnight digest for Kristina to gel extract. Required amounts of crystal violet and calcofluor dye were pipetted into a 96 well-plate as desired. **08/02** Re-made phosphate buffered LB. Excised 5444bp fragment of HindIII/NdeI pMAL digest and gave to Kristina to extract with her kit. Used speedvac DNA concentrator to concentrate the gel extraction products. Julianne and Helia used the 5444bp and pofut2 PCR product to attempt in-fusion construction of our construct (unsuccessful). **08/03** Solutions were prepared but had to be remade since stimuli, LB and phosphate buffer solutions had to all be autoclaved separately (otherwise they would form precipitates). **08/04** **08/05** Started working on safety scenarios with Al. Worked on remaking the motility plates using a 0.3% agar in LB solution instead of TBA with Congo Red and Coomassie Blue. **08/06** Overnight cultures from glycerol stocks were made for all 23 E. coli strains. **08/07** **08/08** Serial dilutions of CF, CV, CR and EtBr planned out. Need to find a cell lysis solution for EtBr since it binds with DNA. Data will be analyzed with Excel. **08/09** Microscope slides were made containing cultures from all 23 strains. The crystal violet and calcofluor dye plates were read on the fluorescence spec. A serial dilution of 1:2 was done across 3 rows of the washed black plate for calcofluor, starting from 1:100 dilution of 1 g/L. A serial dilution of 1:5 of original CR solution was done. Both sets of data were read on the spec in the Houry Lab. Graphs were prepared for the initial CV serial dilution, the recent CR and CF data.Worked on motility assay validation. **08/10** Overnight cultures of MG, BW, (delta)csgD, (delta)fimH and (delta)ompR were made. Worked on using different strains for validating motility assay. **08/11** A 1:2 serial dilution of a 1:100 dilution of 0.1% CV solution was done. Absorbance readings were taken. EtBr protocol was modified. To get the standard dilution curve of EtBr, we placed cells inside and add increasing volumes of ethanol:acetic acid. Measured OD600 for MG1655 and B25113 and dropped 1 microlitre onto the motility plates. **08/12** The EtBr protocol was performed over 4 h. 3 rows had a constant volume and concentration of EtBr, whereas 3 others had a constant of EtOH: acetic acid solution. 3 control wells had Milli-Q, 3 had EtBr and LB, 3 had EtOH: acetic acid and LB. Regular and kan CR plates were made. 2 replicates were made for each type of plate with MG and BW to test whether the glycerol stocks of the WT strains are contaminated. **08/13** Decided that motility assay needs to be repeated by just dipping the pipette tip into the overnight culture. Made a new batch of motility plates and made 70% EtOH. The EtBr protocol was repeated with a 1:10 dilution of 0.5 mg/mL EtBr. CR + BB plates were used to replate glycerol stocks of wild type strains. **08/14** Worked with Al on the safety scenarios guide. Re-made TSB and TB solutions. EtBr protocol was repeated but only for increasing concentrations of EtOH: acetic acid with constant volume of the 1:10 dilution of 0.5 g/L EtBr solution. Non-kan antibiotic plates were made to test contamination of some deletion strains. Insert region for our master plasmid was constructed through PCR extension of two complimentary template primers. In-fusion of this into the 5444bp fragment of HindIII/NdeI pMAL digest to create pEBS (*E. coli* biofilm system) expression plasmid and subsequent transformation of Stellar competent cells was carried out. **08/15** Made Coomassie blue-Congo Red agar for Colony Morphology plates. Analyzed plate reader data. Isolated areas on the graph where the variation of fluorescence with concentration is linear to confirm with another pipetting scheme. Question: does background fluorescence interfere with that of bound EtBr? We need a lysis buffer that will make suspensions clear for the EtBr assay. Dr. Steipe recommends making a TBE-EtBr stock solution to avoid pipetting small volumes. Revision of protocol: should include sodium azide. In-fusion insert was verified to be correct on a 1% TBE agarose gel. **08/16** Prepared MG1655, BW25113, and deletion strains for csgD, csgA, and bcsA with with stimuli. PCR of fim operon, csgBA operon, and bcsA. **08/17** In-fusion assembly of pEBS trial #2. **08/18** No colonies from pEBS assembly trial #2. **08/19** Made sodium azide and culture suspension in TBE. Also made more ethanol: acetone 80:20 solution with anhydrous ethanol and acetone from the fumehood. Results show linear variation of RFU with the concentration of culture (treated with sodium azide.) **08/20** **08/21** Tried sodium azide protocol with 2x and 1/2x EtBr in comparison with 2 days ago. **08/22** **08/23** Made a list of strains to be plated from glycerol stocks on Monday when we have dry ice. Corrected working concentration of Kan to 5 mL/L on wiki based on publications, and grew stimuli cultures thus. **08/24** **08/25** Grew 6 uncontaminated cultures in Phosphate buffered Kan LB and put in shaker. **08/26** **08/27** **08/28** **08/29** In-fusion assembly of pEBS trial #3. Transformed cells plated on ampicilin plates. **08/30** Colonies found from pEBS assembly #3 and LB+Amp overnight cultures made. **08/31** Miniprep of overnight cultures to extract supposed pEBS.Overnight analytical digest using BseRI and ScaI to confirm successful plasmid construction. **09/01** Digested samples ran on 1% TBE agarose gel to confirm successful pEBS construction Preparative digest of successful pEBS using BseRI. This linearized vector will be used to accept target genes amplified by PCR from MG1655. **09/01** **09/02** In-fusion of target genes (ydeH, fimB, csgD, ompA, mlrA) into pEBS. **09/03** Additional analytical digests of pEBS, PCR products from 08/16. pEBS sent for sequencing using M13 primers from TCAG facility at Sick Kids Hospital. 5 in-fusion colonies for each target gene started as overnight cultures. MG1655 and BW25113 overnight cultures started from glycerol stocks **09/04** Chemically competent MG1655 and BW25113 made. Minipreps of in-fusion overnight cultures. Isolated plasmids digested with ScaI and NotI to confirm successful cloning of target genes into pEBS. Gel ran to confirm this. Results negative. Another pEBS linearization/preparative digest conducted. **09/05** Gel ran to confirm that insert/target genes were not degraded and are of correct length. fimB, csgD, ompA, ydeH, and mlrA intact and correct. Fim operon, bcsA, and csgBA were not properly amplified. In-fusion of target genes (ydeH, fimB, csgD, ompA, mlrA) into pEBS trial #2. **09/06** Successful colonies from target genes In-fusion trial #2 started as overnight cultures; 5 separate colonies per gene along with linear backbone control. pEBS sequenced at TCAG and confirmed correct. Assays for (delta)rcsA, (delta)fimA and (delta)csgD were performed. **09/07** Minipreps of pEBS+target genes overnight cultures. Assays for (delta)bcsA, (delta)pgaB and (delta)ompR were performed. **09/08** Digest of the day before’s minipreps using NotI to confirm correct insert’s (target gene’s) presence. 1% TAE agarose gel ran to confirm this. Positive results found for all 5 genes. Assays for (delta)fimA, and (delta)ompA were performed. **09/09** Analytical digest of pSB1C3 to confirm integrity prior to transfer of target genes from pEBS to it for biobrick submission. **09/10** Transformation of MG1655 and BW25113 (via heat shock) with pEBS+target genes. Constructs used to transform sent for sequencing. Gel of pSB1C3 digest confirms it is intact. **09/11** Analytical digests of yesterdays transformation plasmids identify them as being pEBS without target genes. Contamination or human error (using the wrong samples) to be blamed. Sequencing results confirm this. Therefore cannot use recently transformed strains. **09/12** Other samples exist of “successful” pEBS+target gene plasmids. Digest all in preparation of ligating target genes into pSB1C3. Linearize pSB1C3. Digested target genes taken from pEBS+target genes constructs confirmed to be of correct size by gel electrophoresis. Ligation of target genes into pSB1C3 and subsequent transformation into DH5 α competent cells. **09/13** Colonies found from ligation reactions (target genes into pSB1C3) and 10 overnight cultures started per gene to allow screening for false positives. **09/14** **09/15** Minipreps of ligation overnight cultures and subsequent analytical digests. Samples digested to screen for false positive ligations. Minimum one found per gene and prepared for submission to the registry. Electrocompetent MG1655 and BW25113 prepared and transformed with pEBS+ (csgD/ydeH/ompA/fimB/mlrA) to be used in assays to characterize new biobricks under lac promoter control. Glycerol stocks of pEBS+target genes and pEBS in stellar E. coli made. **09/16** pSB1C3+biobricks (target genes) shipped to the registry and sent for sequencing. **09/17** **09/18** **09/19** 0.3 g/L stock of IPTG in buffered LB. It was induced at 0.3 mM in each well at the beginning of stimuli inoculation. The Biobrick inserts (mlrA, ompA, csgD and fimB) were transformed into BW previously. Overnight cultures of these were inoculated with various stimuli. **09/20** BW-WT and MG-WT were inoculated along with various stimuli. **09/21** Overnight culture of BW-Y (containing ydeH insert) was inoculated with various stimuli. The BW strains containing various Biobrick inserts that were inoculated 2 days earlier were assayed. **09/22** Amount of agar plate ingredients were calculated for “media” plates (e.g., TB plates, TSB plates). **09/23** BW and MG Wild type strains were assayed. **09/24** Colony morphology plates were poured for various media. Overnight cultures of BW with Biobrick inserts were made. **09/25** Overnight cultures made the day before were streaked onto dried colony morphology media plates. **09/26** pSB1C3 biobrick constructs sequencing results analyzed. fimB and ompA biobricks appear to have mutations and require further investigation. **09/27**

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