Team:Toronto/Notebook

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.

05/29
Learned how to use the incubator with shaker and how to operate the Moraes lab Thermocycler and Spectrophotometer (details uploaded to Wiki). Also investigated the ideal rbs sequence for our genes. According to From Genes to Clones, the rbs should be 4-9bp upstream of ATG. It's stronger if you add adenines upstream of the RBS and it comes before minor genes in an operon. If we're interested in operon expression we must keep the stoichiometric balance of each minor gene by preserving the native rbs sequences.

05/30
Project Milestones are established:
1. Primer design with pMal vector.
2. Amplification and cloning of genes
3. Establishing selection and cultivation conditions with assays and stimuli
Sub-goals within each step were established as well. Prepared glycerol for inoculation and streaking. Lab members were assigned stimuli/assays to do and today we made a list of the ingredients from these protocols which we didn't have. Designed primers for CsgD after establishing RBS and other sequences.

06/01
Crystal violet assay – Discussed the different procedures for this with Afiq and Seemi.

06/02
First round of crystal violet protocols established.

06/03
Edited primers.

06/05
New cloning strategy introduced by Kristina: InFusion by Clontech. Worked to redesign primers for this purpose.15bp homology required between each insert. Prepared M63 minimal media.

06/06
Made a culture of MG1655 from a plate Michael brought over from BioZone for the purpose of making competent cells. Edited primers.

06/07
Learned to determine OD600 of growing MG culture by diluting. Made agar and 10% glycerol from 40% stock. Poured plates and streaked the little tabs that the deletions came in on top of each plate by suspending in sterile LB medium. Strains were incubated at 7 PM.

06/09
Made liquid cultures of each deletion strain so we can have glycerol stocks. Got trained on microscope.

06/12
In order to wrap up primer design, Dr. Steipe instructed us on how to make the RBS overlap, checked over the PCR product schema, and suggested a double stop codon. Re-established goals for the project and reviewed stimuli and assays. Booked equipment training with Artur in the Division of Teaching Labs so we can start trying the ELIZA and spectrophotometer downstairs.

06/13
Made a list of strains to be tested for biofilm proteins. Incubated MG1655 at room temperature without shaking. Prepared for crystal violet assays with ethanol, temperature and salt stimuli on clear plates

06/14
Artur trained us to use the ELIZA and Titrek Multiscan spectrophotometer. Made stock solutions of NaCl+LB at varying concentrations, 50mM-350mM.

06/16
Pairing Stimuli with Assays – Organized a chart of stimuli variations matched with Assays for the different proteins of interest. Ran a sample crystal violet with MG1655 and realized that readings are not noticeably different from each other. Made 400g/L PEG solution for osmolarity stimuli and ordered ingredients for yeast media. Grew WT cultures overnight at 250 RPM.

06/17
Pipetted CV plates with varying salt concentrations. Made more salt-free medium and made stock CR. Amount of ethanol that needs to be pipetted for each stimulus treatment was calculated for 100% ethanol. The amount didn't work, since less than 1 microL was required for some. Thus, 70% ethanol was decided upon. Plate maps were drawn for osmolarity (using PEG-4000) and ethanol stress stimuli. CR solution was made.

06/18
Prepared ethanol assay with MG1655 and BW25113. Prepared LB agar plates for colony morphology assay. Prepared yeast media (YPD) and 20% glucose solution for time to yeast agglutination assay.

06/19
To prepare for CR assay, we diluted overnight culture to OD600=1 and did the CR protocol with the prepared culture. OD480 absorbance was taken for BW and MG.

06/20
The PEG & Ethanol crystal violet assay was conducted again.

06/21
Made liquid overnight cultures of E. coli MG1655 and BW25113.

06/22
Inoculated motility and Colony morphology plates. Made wet mounts of cultures to observe yeast (which seem to aggregate at bottom of tube). Made more YPD. Having trouble getting the spec to make sensible OD600 measurements when diluting culture.

06/23
Prepared a 1:100 dilution of overnight culture and made motility plates. Used microscope to visualize bacteria using Koehler illumination. Made notes on colony morphology results i.e. the criteria on which to judge the colonies e.g. form, elevation, etc. Dr. Steipe suggests to validate CV by measuring OD600 throughout the growth stages, tapping hard into the bench for the washing step, putting more CV inside, and randomizing wells. He also suggests a swarm plate recipe with 'liquid agar'. We must first dilute our samples to OD<1 as the instrument is less accurate at high optical densities.

06/24
Meeting: CV protocol was improved on and data from the absorbance readings was analyzed using Excel. Looked up protocols for Ag43 assay. Yeast were obtained from the Meneghini lab and streaked onto YPD plates, then incubated O/N. The protocol for time to yeast agglutination was copied down.

06/25
Prepared swarm/motility plates with tryptone broth agar (TBA). Prepared motility assay samples for BW25113.

06/26
Prepared frozen stocks of yeast by inoculating them. Prepared swarming agar for motility assays. Pipetted another stimuli plate using OD600 = 1 cultures. Prepared cellulose assay (brilliant blue and congo red) plates. Made glycerol stocks of E. coli with pKD46, pKD3.

06/27
Incubated plates for motility assay, prepares OD600=1 culture for yeast agglutination assay, and repeated CR assay. Finished pipetting CV sodium stimuli plates at 10 PM and covered from light; grew overnight cultures. The colony morphology assay was performed. The colony morphology assay protocol was written out. A fresh 96-well plate was pipetted into according to a new plate design for the osmolarity and sodium assay. M9 salts have NaCl, but the effects are likely small of the amount of salt present in M9 salts in regular LB.

06/28
Made OD600=1 cultures of bacteria and subcultured yeast on YPD (still not cloudy). Did CR and CV assay protocols and re-inoculated motility plates. Afiq contacted Lynne Howell about a budget-friendly PGA assay.

06/29
Did CV assay on osmolarity, sodium and ethanol plates. Assayed for agglutination phenotype with yeast cells but there doesn't seem to be much happening when bacteria and yeast mix. Measured motility haloes of the two WT strains, which seem to indicate that BW has smaller motility radius than MG, and then returned the plates to darkness for 24 hours.

06/30
Finished assay on sodium plates but there were some issues:
1. Having the lid on may not be a good idea. It fits the sterile and non-sterile plates differently.
2. Colour trend of resolubilized CV doesn't seem to follow that of the plate we pipette from.
3. Amount of liquid remaining in the plate that we pipetted from is not uniform at least partially due to the odd way tips stick to the multipipettor.
4. Does this assay measure biofilm or the lack of it?

07/02
Data on empty 96- well plate with and without lid were taken from the 96-well plate reader.

07/03
TBA plates were made, OD = 1 cultures were made, overnight cultures and sodium-less LB cultures were made for wild type strains.

07/04
Overnight cultures were prepared with regular LB and sodium-less LB. TBA plates were also poured.
Started working on establishing stimuli protocols.
A meeting was held in preparation for the iGEMxDIYBio talk, and a general outline was prepared.

07/05
Researched an adhesion assay that would pair with crystal violet for quantification. Plated competent cells on LB agar, left to grow at 37C for use in colony PCR.

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