IGEM:PennState/Labbook/LucienWeiss

Lucien Weiss Lab Notebook

• Penn State iGEM 2005-2007
  • [iGEM 2005]
  • [iGEM 2006]
  • [iGEM 2007]
• Penn State Undergraduate
• Email luw134@psu.edu
• Phone (814) 880 5909

March 12, 2007: Motility Assay

Experiment Aims

Test motility on a variety of surface hardnesses in order to determine optimal level to use for assays.

Procedure

Eiken Agar Plates were made using a modified version of general procedure 1. Increased agar concentrations were made to increase hardness of gel and reduce the speed of motility.

.20% Eiken Agar Plates:

• .25g Bactotryptone
• .20g NaCl
• .15g sodium citrate
• .05g Eiken Agar (for 20EikenAgar)

.30% Eiken Agar Plates:

• .25g Bactotryptone
• .20g NaCl
• .15g sodium citrate
• .075g Eiken Agar (for 30EikenAgar)

.40% Eiken Agar Plates:

• .25g Bactotryptone
• .20g NaCl
• .15g sodium citrate
• .10g Eiken Agar (for 40EikenAgar

1. Constructs were transformed into RP3087
   • YFP Sender Cells-Sender (LVA+)-RP3087 w/ [I14032+B0030+C0161 A3]+E0430
   • Sender Cells-Sender-RP3087 w/ I14032+B0030+C0161 (B2)
   • Receiver-RFP+Receiver-RP3087 w/ I13321+[J09855+B0033+J09274]
2. Grown overnight at 37 degrees C on 50 micromolar Kan LB plates
3. 3mL cultures inoculated off of the plate and grown at 30 degrees C until OD590~.45
4. 3uL placed of receiver cells placed in various distances from 3uL inoculated sender colony.

Results:

.20% Eiken Agar Sender and Receivers
.30% Eiken Agar with sender and receiver cells after 8 hours
.30% Eiken Agar Sender and Receivers
.30% Eiken Agar with sender and receiver cells after 8 hours	.30% Eiken Agar with regular sender and receiver cells after 20 hours
.30% Eiken Agar with Degradation Tagged AHL
.30% Eiken Agar with sender produced AHL tagged for degradation and receiver cells after 8 hours	.30% Eiken Agar with sender cells producing AHL tagged for degradation and receiver cells after 20 hours
.40% Eiken Agar Sender and Receivers
.40% Eiken Agar with sender produced AHL tagged for degradation after 8 hours	.40% Eiken Agar with regular sender and receiver cells after 8 hours

Observations:

.20% agar plate difficult to handle as agar was extremely fragile. Results were diminished by heavy diffusion. .30% agar results in fair motility and is much easier to handle .40% agar results in no motility

Conclusions:

Future Experiments:

Retry .20% Agar experiments with set distances away from sender Experiment with temperature changes

March 13, 2007: Motility Assay

Experiment Aims

Test induced motility at a variety of distances from sender cells.

Procedure

Eiken Agar Plates were made using a modified version of general procedure 1. Agar concentrations were kept at .20%

.20% Eiken Agar Plates:

• .25g Bactotryptone
• .20g NaCl
• .15g sodium citrate
• .05g Eiken Agar (for 20EikenAgar)

1. Constructs were transformed into RP3087
   • YFP Sender Cells-Sender (LVA+)-RP3087 w/ [I14032+B0030+C0161 A3]+E0430
   • Sender Cells-Sender-RP3087 w/ I14032+B0030+C0161 (B2)
   • Receiver-RFP+Receiver-RP3087 w/ I13321+[J09855+B0033+J09274]
2. Grown overnight at 37 degrees C on 50 micromolar Kan LB plates
3. 3mL cultures inoculated off of the plate and grown at 30 degrees C until OD590~.45
4. 3uL placed of receiver cells placed in various distances from 3uL inoculated sender colony.

Results:

.20% Eiken Agar Sender and Receivers
.20% Eiken Agar with sender and receiver cells after 8 hours	.20% Eiken Agar with sender and receiver cells after 8 hours

Observations:

Difficult to discern motility on .20% Eiken Agar plates

Conclusions:

Future Experiments: Increase Agar Concentration Experiment with temperature changes

March 16, 2007: Motility Assay

March 29-April 1, 2007: Institute of Biological Engineering

Poster: Bacterial Relay Race

IBE Poster

April 22, 2007: Project Proposal Presentations

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Project Proposal: Pressure Sensing in Bacteria

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Project Proposal: Bi and Sept Stable Oscillator

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April 28, 2007: Synthetic Biology 3.0

Biology 3.0 Website

May 15, 2007: General Procedures

Innoculation from plate

Culture Dilutions EX 1-1:100 Dilution

1. Add 3mL fresh LB to a new culture tube
2. Add 3uL of appropriate (1:1000)antibiotic solution
3. Take 30uL from starter culture and place in fresh media.
   • Becareful to not touch the sides of the tube with the pipetmen.

Checking cell OD. Using Spectrophotometer

1. Turn on spectrophotometer (switch on the back to the right)
2. Allow the startup to run, and then turn off deuterium Light (needed at hv <300nm)
3. Turn wavelength to 590nm
4. Zero with 1mL LB in Cuvette
5. Do an appropriate dilution (suggested 1:5, 1:10) of culture with fresh LB into a fresh Cuvette

note 1: if you are trying to find out how long it will be until cells are at OD .6 (mid log) you can take the OD at two seperate times and use http://molbiol.edu.ru/eng/scripts/01_02.html. note 2: diluting a culture down to OD .6 will not put it in mid log phase; dilute down to .15 and allow it to grow back up to .6 if your culture has grown too long.

Miniprep

1. Spin down 3mL of saturated cell culture, remove supernatant
2. Resuspend pelleted bacteria in 250uL Buffer P1 and transfer to a 1.5mL micro centrifuge tube.
3. Add 250uL Buffer P2 and invert the tube gently 4-6 times to mix.
4. Add 350uL Buffer N3 and invert the tube immediately but gently 4-6 times.
   • the solution should be come cloudy
5. Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge
   • allign tubes such that the cap clip is facing outward
6. decant supernatant into spin column
7. Centrifuge for 60s. Discard flow-through.
8. Add .75mL Buffer PE and centifuge for 60s.
9. Discard flowthrough and centrifuge for 60s
10. Place the spin column into a new 1.5mL microcentrifuge tube.
11. Add 30-50uL of water (8.5>pH>7) to the center of each spin column.
12. Let tubes stand vertically for 1min, then centrifuge for 1min.

10uL Digest

1. 2.5uL H2O
2. 4.0uL Vector
3. 0.5uL Insert
4. 1.0uL Buffer X
5. 1.0uL BSA
6. 0.5uL Enzyme 1
7. 0.5uL Enzyme 2

20uL Digest

1. 2.0uL H2O
2. 11.uL Vector
3. 2.0uL Insert
4. 2.0uL Buffer X
5. 2.0uL BSA
6. 0.5uL Enzyme 1
7. 0.5uL Enzyme 2