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Experiments & Protocols

UV kill curve experiments

Initial pilot test

UV box (ExoTerra flourescent bulbs and LEDs from nail curing light; Insulfoam sides) was set up in flow hood w/ 8 inch tall rack approximately centered. A horizontal reflector was placed on top of the rack.

Before start, two plates had been prepared for the experiment: one 24-well culturing plate containing 100uL of liquid culture of transformed pGLO HB101 and untransformed HB101 (grown overnight in TSB at 37oC w/ shaker) in each of 12 wells, and one 96-well plate w/ 90uL of TSB pr. well in 48 wells for dilution.

The culturing plate was used for UV exposure of the cultures. At intervals of 1, 2, 5, 10, 20, 30 mins, the plate was removed from the box, and 10uL of sample was removed from each of 2 wells (1 for each strain). The sample was transferred to a well on the dilution plate to create an initial 10-fold dilution, and the culturing plate was returned to the UV box for further exposure.

From the initial 10-fold dilution, further dilutions (10-2, 10-3 and 10-4) were created. For each dilution, 4 drops of 10uL were spotted onto one quadrant of a petri plate (LB agar); plates were then incubated for approx. 36hrs at 37oC, and colonies were counted.

First experiment, Aug. 27th, 2015

UV box (ExoTerra fluorescent bulbs and fluorescent nail curing light; Insulfoam sides) was set up in flow hood w/ 8 inch tall rack approximately centered.

Before start, plates had been prepared for the experiment: one 24-well culturing plate containing 100uL of liquid culture of transformed pGLO HB101 and untransformed HB101 (grown overnight in TSB at 37oC w/ shaker) in each of 12 wells, and four 96-well PCR plate w/ 90uL of TSB pr. well in 60 wells for dilution.

The culturing plate was used for UV exposure of the cultures. At intervals of 10, 20, 30, 40, 50, and 60 mins, the plate was removed from the box, and the full 100uL sample was removed from each of 2 wells (1 for each strain). The samples were transferred to 2 wells on the dilution plates, and the culturing plate was returned to the UV box for further exposure.

From the exposed samples, a dilution series (10-1, 10-2, 10-3,10-4, and 10-5) were created. For each of the 100- to 100000-fold dilutions, 6 drops of 2uL were spotted onto one quadrant of a petri plate (LB agar; arabinose added for HB101+pGLO) using a 10uL multichannel pipette; plates were then incubated for approx. 36hrs at 37oC, and colonies were counted as an average of each of 6 spots.

Second experiment, Sept. 6th, 2015

Liquid cultures of transformed pGLO HB101 and untransformed HB101 (grown overnight in TSB at 37oC w/ shaker) were prepared by transferring 1.8mL of culture to a microcentrifuge tube and spun for approx. 5 min at 3000 rpm. The supernatant was removed, and 1.8mL of phosphate-buffered saline (PBS) was added to each tube and vortexed gently to resuspend cells. This washing procedure was repeated 3 times. Finally, cells were resuspended in PBS to approx. OD600 = 0.8.

UV box (ExoTerra fluorescent bulbs and fluorescent nail curing light; Insulfoam sides) was set up in flow hood w/ 10.5 inch tall plastic contained placed upside-down approximately centered under the lights, and a horizontal reflector was placed on top of the container. The lights were turned on 5 mins prior to start to ensure that the fluorescent tubes had sufficient time to warm up. Combined UV-A and UV-B light intensity was measured at approx. 1.75mW/cm2, and temperature was 32oC.

1.8mL of each cell resuspension was transferred to two 35mm petri dishes; before starting UV exposure, 3*10uL were transferred to three wells on a 96-well PCR plate w/ 90uL of PBS pr. well. The petri dishes were then placed on the horizontal reflector under the UV lights, and the timer was started. At intervals of 10, 20, 30, 40, 50, and 60 mins, the petri dishes were removed from the box, and 3*10uL samples from each dish were transferred to wells on the dilution plates. The dishes were then returned to the UV box for further exposure.

From the exposed samples, a dilution series (10-1, 10-2, 10-3,10-4, and 10-5) were created. For each of the 100- to 100000-fold dilutions, 6 drops of 2uL were spotted onto one quadrant of a petri plate (LB agar; arabinose added for HB101+pGLO) using a 10uL multichannel pipette; plates were then incubated for approx. 20hrs at 37oC, and colonies were counted.

Third experiment, Sept. 7th, 2015

Liquid cultures of transformed pGLO HB101 and untransformed HB101 (grown overnight in TSB at 37oC w/ shaker) were prepared by transferring 1.0mL of culture to a microcentrifuge tube and spun for approx. 5 min at 3000 rpm. The supernatant was removed, and 1.5mL of phosphate-buffered saline (PBS) was added to each tube and vortexed gently to resuspend cells. This washing procedure was repeated 3 times. Finally, cells were resuspended in PBS to approx. OD600 = 0.35.

UV box (ExoTerra fluorescent bulbs and fluorescent nail curing light; Insulfoam sides) was set up in flow hood w/ 10.5 inch tall plastic contained placed upside-down approximately centered under the lights, and a horizontal reflector was placed on top of the container. The lights were turned on 5 mins prior to start to ensure that the fluorescent tubes had sufficient time to warm up. Combined UV-A and UV-B light intensity was measured at approx. 1.96mW/cm2, and temperature was 32oC.

1.5mL of each cell resuspension was transferred to two 35mm petri dishes; before starting UV exposure, 2*10uL of each strain were transferred to wells on a 96-well PCR plate w/ 90uL of PBS pr. well. The petri dishes were then placed on the horizontal reflector under the UV lights, and the timer was started. At intervals of 15, 30, 45, 60, 75, and 90 mins, the petri dishes were removed from the box, and 2*10uL samples from each dish were transferred to wells on the dilution plates. The dishes were then returned to the UV box for further exposure.

From the exposed samples, a dilution series (10-1, 10-2, 10-3,10-4, and 10-5) were created. For each of the 100- to 100000-fold dilutions, 6 drops of 2uL were spotted onto one quadrant of a petri plate (LB agar; arabinose added for HB101+pGLO) using a 10uL multichannel pipette; plates were then incubated for approx. 20hrs at 37oC, and colonies were counted.

Bacterial Transformation Experiments

Transformation of HB101 and CFSC #3600 Bacteria with Shinorine Producing Genes, Sep. 1st-Sep. 3rd, 2015

We transformed 2 different strains of bacteria (HB101 and CGSC #3600) and inserted the pET29b plasmid (with shinorine producing genes).

Materials

-HB101 E. Coli strain

-CGSC #3600 E. Coli Strain (mutagenic strain from the Yale Coli Genetic Stock Center http://cgsc.biology.yale.edu)

-LB Media

-75mM CaCl2

-pET29b plasmid backbone (Ava3858-3855, Ava3858-3856, Ava3858-3857 are genes of interest)

-pGLO plasmid

-kanamycin (50ug/ml)+ampicillan (100ug/ml) plate

Protocol

We ran transformations with the following plasmids for each E. coli strain.

1. pET29b-Ava3858-3855 (Kan plate)

2. pET29b-Ava3858-3856 (Kan plate

3. pET29b-Ava3858-3857 (Kan plate)

4. pGLO (+control) (Amp plate)

5. no plasmid (-control) (Amp plate)

Competent Cell Preparation:

1. Pick single colony from plate stored at 4 oC.

2. Innoculate colony into 3 ml of LB in a 15 ml culture tube

3. Incubate at 37oC with shaking ~ 16 - 24 hours (overnight)

4. Add 1ml of the overnight culture to 10 ml fresh LB in a 50 ml Falcon tube(provides more aeration than a 15 ml tube but not as much as an Erlenmeyer flask

5. Incubate @ 37oC with shaking 2.5 – 3.5 hours - The culture will be in log phase and turbid

6. Centrifuge at 3,500 RPM for 10 mins (big floor centrifuge) - balance w/water-filled Falcon tube or use the two tubes as prepared in step 4 to balance each other.

7. Decant supernatant and re-suspend in 2 ml sterile ice cold 75mM CaCl2 (11 g CaCl2•2H2O/100 ml water) CaCl2•2H2O MW = 147.01

8. Centrifuge at 3,500 RPM for 5 mins (big floor centrifuge)

9. Decant supernatant and re-suspend in 1 ml sterile ice cold 75mM CaCl2

Transformation:

10. Put 50 μL of cell solution into PCR tubes ON ICE

11. Add DNA (1-2 μL plasmid DNA or 10-20 μL PCR product) to 50 μL of cells and run in thermal cycler on program TRANS: (30 min 4 ̊C, 45 sec 42 ̊C, 5 min 4 ̊C)

12. While thermocycler is running, fill sterile 1.5 ml microcentrifuge (Eppi) tubes with 450 μL sterile LB (1:10 dilution of transformed cells), and pre warm in water bath to 37

13. After thermocycler is finished, transfer the 50 μL of transformed cells into pre-warmed 450 μL LB Eppi tube to yield a total of 500 μL in each eppi tube

14. Incubate 45 min-1hr at 37oC with shaking

15. Centrifuge grown out cells at 12,000 RPM for 30 sec - 1 min.

16. Decant most of the supernatant--leave about 150-200 μL.

17. Resuspend cells in this solution

18. Transfer cells onto solid growth media

19. Incubate plates at 37 ̊C, look for colonies the next