Team:Pittsburgh/Notebook

 






Notebook

 

Virtual meetings conducted to conceptually develop our project, as the University of Pittsburgh research operations were remote until 07/24/2020.

Took inventory of the lab.

Lpp-OmpA Streptavidin Transformation Attempt #1: plates did not have any colony growth, likely because it was only grown with ampicillin as antibiotic.

Lpp-OmpA Streptavidin Transformation Attempt #2: the transformation of plasmid was successful due to growth on the non-antibiotic plate; however we require another attempt to figure out what antibiotics this plasmid is resistant to.


Table 1. 8/4/20 Transformation results of plasmids Bba_J36848 and PVS0043 after overnight growth at 37°C

Cell Plasmid Plate Media + Antibiotic Growth
DH5alpha Bba_J36848 LB + no antibiotic Lawn/film formed
DH5alpha Bba_J36848 LB + chlor No
DH5alpha Bba_J36848 LB + AMP No
DH5alpha Bba_J36848 LB + Kan No
DH5alpha PVS0043 LB + AMP Yes. Two small colonies formed

Figure 1. Transformation results of plasmid Bba_J36848 (streptavidin) after overnight growth at 37°C

Lpp-OmpA Streptavidin Transformation Attempt #3: unclear results because of a mistake made with the protocol (parafilm applied to plates when growing overnight).

Table 2. 8/10/20 Transformation results of plasmid Bba_J36848 after overnight growth at 37°C

Cell Plasmid Plate Media + Antibiotic Growth
DH5alpha Bba_J36848 LB + no antibiotic Lawn/film formed
DH5alpha Bba_J36848 LB + chlor No
DH5alpha Bba_J36848 LB + AMP No
DH5alpha Bba_J36848 LB + Kan No

Lpp-OmpA Streptavidin Transformation Attempt #3 (continued): Removed the parafilm from the plates to regrow again overnight; found that the backbone of the streptavidin plasmid is resistant to chlor.

Table 3. 8/11/20 Transformation results of plasmid Bba_J36848 after night at 37°C

Cell Plasmid Plate Media + Antibiotic Growth
DH5alpha Bba_J36848 LB + no antibiotic Lawn/film formed
DH5alpha Bba_J36848 LB + chlor Yes, one large and one smaller colony formed
DH5alpha Bba_J36848 LB + AMP No
DH5alpha Bba_J36848 LB + Kan No

Figure 2. Transformation plates from 9 AddGene plasmids grown in DH5alpha

From the transformation confirmation plates of 9 AddGene plasmids in DH5alpha, and a streaked streptavidin plate, 11 total liquid cultures were made. The streptavidin cultures did not grow, and the 9 DH5alpha constructs were made into freezer stocks.

Attempt #2 at growing liquid cultures from the streptavidin plasmid, but they did not grow.

Attempt #3 at growing liquid cultures for streptavidin and attempted to miniprep the culture for transformation, however it did not grow. Attempt #4 of the streptavidin liquid cultures were set to grow overnight.

Miniprepped streptavidin from overnight liquid cultures, and attempted transformations of AddGene plasmids into BL21 competent cells and streptavidin into BL21 and DH5alpha competent cells.

Transformation plates were checked, however all attempts were unsuccessful.

Attempt #2 of AddGene plasmid transformation into BL21 competent cells.

Figure 3. 8/25/20 Transformation Plate Results of 9 AddGene plasmids grown in BL21

Made liquid cultures from colonies on transformation plates of AddGene plasmids into BL21 cells. Made freezer stocks from those liquid cultures. Additionally, transformed streptavidin into DH5alpha and BL21 competent cells, and plated overnight.

Figure 4. 8/26/20 Transformation Plate Results of streptavidin plasmids grown in BL21 and DH5alpha

After realizing nothing of value grew on the streptavidin transformation plates from 8/26/20, new liquid cultures were made of streptavidin. These cultures were miniprepped, transformed into DH5alpha and BL21 competent cells, and plated overnight.

Liquid cultures of the 9 AddGene constructs were prepared and grown overnight.

Overnight liquid cultures of the 9 AddGene constructs (prepared on August 31) were induced for protein expression today

Liquid cultures of the GFP constructs (SW8765-Rosetta, and WSC1068-8W25113) were prepared and grown overnight.

From the overnight cultures of Rosetta and WSC1068, freezer stocks were made, and another round of overnight cultures (for the same constructs) were set up to express the proteins tomorrow.

Inoculated and induced liquid cultures for expression of the 9 AddGene plasmids (using the overnight cultures prepared yesterday). Also ran a heating assay for the expressed constructs that were stored in –80 freezer.

Prepared liquid cultures and the PCR machine for heat assay experiment.

Attempt #2 at growing liquid cultures from the streptavidin plasmid, but they did not grow.

Attempt #3 at growing liquid cultures for streptavidin and attempted to miniprep the culture for transformation, however it did not grow. Attempt #4 of the streptavidin liquid cultures were set to grow overnight.

Miniprepped streptavidin from overnight liquid cultures, and attempted transformations of AddGene plasmids into BL21 competent cells and streptavidin into BL21 and DH5alpha competent cells.

Transformation plates were checked, however all attempts were unsuccessful.

Liquid cultures grown to make more competent cells.

Liquid cultures were grown at 30 degrees Celsius from 1:40pm-6:45pm and left to grow further overnight in preparation for the Temperature Sensitive Dimer Induction experiment (measuring GFP expression). OD600 was measured in 1.5 hour intervals after induction start time. Last measurement was taken at 6:45pm (OD 0.018).

We transformed constitutively mCherry expressing cells with the heat sensitive dimers.

Re-plated the transformed bacteria on LB-Agar plates to confirm that the transformation was successful. Left overnight to incubate and grow at 37 degrees Celsius.

WSC1068 cells were transformed with heat-sensitive constructs after being incubated at 37°C overnight on LB-AMP/KAN plates. Liquid cultures were grown from colonies on plates and made into freezer stocks.

Figure 5. Shows the transformation results of WSC1068 cells with nine heat-sensitive constructs after incubating at 37°C overnight. Colonies were growing on each LB-AMP/KAN plate. Single colony was selected for liquid culture and preparation of freezer stocks

Binding amine-coated magnetic nanoparticles (amine-MNP) to E. coli competent cells by suspending in solution.

Table 4. OD change before and after addition of MNPs

Initial OD600 (no MNP added) Final OD600 (MNP added and removed)
DH5alpha Bba_J36848 LB + no antibiotic
Sample 1 7.006 5.874
Sample 2 1.886 0.561
Sample 3 0.411 0.608
Sample 4 (no cells, control) 0.202 (blank) 0.209 (blank)

Figure 6. Separation of Amine-MNP Capture on 10/13; shows the amine-MNP suspended in E. coli solution. Based on visual observation, there was no clear magnetic separation between supernatant and MNPs. The supernatant extracted for absorbance measurement was cloudy and likely contained MNP

Attempt #2 of binding amine-coated MNPs to E. coli competent cells.

Figure 7. Separation of Amine-MNP Capture (Sample 1-3) on 10/14/2020