Day 1, Monday 11/06

People In Lab:Amalia, Carla, Daniel, Jasmeen, Nina, Tashi

Agenda:

• Transform 2J interlab plasmid into DH5α and plate on LB+CAM

• LB+CAM plates

• Interlab calibration #2: Particle Standard Curve - Microsphere Protocol

• Electroporate MG1655ΔlacI

• Transform pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI

• Make and autoclave PBS for interlab calibration #3

• Autoclave double distilled water

Equipment used:

• Autoclave

• Bunsen burner

• Plate reader

• Thermomixer

• 37°C incubator in room 301

• Electroporator

Protocol: iGEM protocols used for transformation, electroporation, LB+agar. Interlab protocol used for Interlab calibration #2: Particle Standard Curve - Microsphere Protocol.

Transformation of 2J plasmid

1μLof 2J plasmid used, 50μLof cells plated

Interlab calibration #2

• 200μLbead stock in wells A1, B1, C1, D1

• 100μLdouble distilled water in wells A2-A12, B2-B12, C2-C12, D2-D12

• performed serial dilution by transferring 100μLeach time, until well A11, B11, C11, D11

• used the plate reader to shake the plate at an amplitude of 3.5 for 7 seconds, and measure the absorbance at wavelength 600nm

Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI

• 2μLof pSB1C3+RFP(10pg/μL)

• 1μLofpET28a+arg1

• plated 100μLon LB+CAM+KAN

• plated 200μLon LB+CAM+KAN

• plated 300μLon LB+CAM+KAN

• plated negative control (100μLof untransformed MG1655ΔlacI)

Next Steps:

• Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• Interlab cell measurement protocol

Day 2, Tuesday 12/06

People In Lab: Amalia, Carla, Daniel, Nina

Agenda:

• Second attempt at pSB1C3+RFP and pET28a+arg1 double transformation in MG1655ΔlacI, testing for incompatibility

• Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• Make overnight cultures from interlab transformation plates

Equipment used:

• Bunsen burner

• Plate reader

• Electroporator

• Shaker in room 315

• 37°C incubator in room 301

Protocol: iGEM protocols used for transformation by electroporation. Interlab protocol used for for Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI (electroporation)

Hypothesis: If we get growth on the positive control, LB+KAN, and LB+CAM plates with transformed cells, but not the double antibiotic plate, we can say that either the plasmids are incompatible, or the double antibiotic plate concentration is too high.

• 1.5μLof RFP (50pg/μL)

• 1.5μLof pET28a+arg1

• plated 150μLof transformed cells on LB+CAM+KAN

• plated 100μLof transformed cells on LB+CAM

• plated 100μLof transformed cells on LB+KAN

• plated 10μLof untransformed cells on LB+KAN (negative control)

• plated 10μLof untransformed cells on LB (positive control)

Interlab calibration #3

• serial dilution was performed and the plate was read

• shaken 5s at amplitude = 2

• excitation = 485nm

• emission = 525nm

• optimal gain used = 75

• read from bottom of plate

• The graph obtained from this experiment indicated an error, will repeat tomorrow

Interlab transformation overnight cultures

• 16 tubes with 5mL of LB and 5μLCAM

• 2 colonies were taken from each of the 8 plates (2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P)

• tubes placed in the shaker overnight at 37° at 250rpm

Observations:

Double transformation of pSB1C3+RFP and pET28a+arg1 into MG1655ΔlacI

• no colonies on negative control plate

• no colonies on 100μL, 200μL, or 300μLplates

100μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI (negative control) on LB+CAM+KAN

100μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN

200μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN

300μL double transformation of pSB1C3+RFP (10pg/μL) and pET28a+arg1 in MG1655ΔlacI on LB+CAM+KAN

Potential reasons for results:

• antibiotic concentration too high (50μL/mL KAN + 25μL/mL CAM)

• MG1655ΔlacIcells may no longer be competent

• left MG1655ΔlacIcells thawing in the ice for too long

• electroporation might have failed

• plasmids may not have been compatible

• we could try sequential transformation (still have to consider antibiotic concentration of LB+KAN+CAM plate)

Next Steps:

Finish Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

Cell growth, sampling, assay (interlab cell measurement)

Day 3, Wednesday 13/06

People In Lab:Amalia, Carla, Daniel, Nina

Agenda:

• Redo Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• Autoclave glass bottles, beads, tips, eppendorf tubes

• Reculture the overnight cultures from the interlab transformation

Equipment used:

• Autoclave

• Bunsen burner

• Plate reader

• 37°C shaker in room 315

Protocol: Used interlab protocol for interlab calibration.

Second attempt at Interlab calibration #3: Fluorescence standard curve - Fluorescein Protocol

• serial dilution was performed and the plate was read

• excitation = 485nm

• emission = 525nm

• optimal gain used = 90

• read from bottom of plate

Reculturing the overnight cultures from the interlab transformation

• 16 tubes with 5mL of LB and 5μLCAM

• 100μLfrom each previous overnight culture placed in respective new tube (DH5α)

• tubes slightly unscrewed and tape placed on lid for aeration in shaker

• tubes placed in the shaker overnight at 37°C at 250rpm

Observations:

Second double transformation pSB1C3+RFP and pET28a+arg1in MG1655ΔlacI

• LB+CAM+pSB1C3+RFP and pET28a+arg1

  • sparsely populated pink colonies

  • presence of some colonies

• LB+KAN+pSB1C3+RFP and pET28a+arg1

  • no visible colonies

• LB+KAN+CAM+pSB1C3+RFP and pET28a+arg1

  • no visible colonies

• LB+KAN+nontrasformed cells (negative control)

  • no visible colonies

• LB+nontransformed cells (positive control)

  • growth on entire plate

Second double transformation pSB1C3+RFP and pET28a+arg1 in MG1655ΔlacI

Potential reasons for these results:

• colonies on LB+CAM+pSB1C3+RFP+pET28a+arg1) because the cells are still competent and were transformed with pSB1C3+RPF

• no colonies on LB+KAN+pSB1C3+RFP and pET28a+arg1not because the cells are not competent, but rather the transformation (pET28a+arg1into MG1655ΔlacI) failed

• no colonies on LB+KAN+CAM+pSB1C3+RFP+pET28a+arg1 because the backbones are not compatible

• no growth on LB+KAN+non transformed cells (negative control) because there was no antibiotic resistance gene in the cells

• we had growth on LB+non transformed cells (positive control) because the cells were viable and there was no selection in the plate against them

  • pET28a = KAN resistant - incompatible with pSB1C3 by group check

  • pSB1C3 = CAM resistant - higher copy number than pET28a

Next Steps:

Finish interlab cell measurement protocol

Day 4, Thursday 14/06

People In Lab:Ahmed, Amalia, Daniel, Monica, Nina, Tashi

Agenda:

• Interlab cell measurement protocol

• Autoclave P1000 tips

• LB media

• LB+CAM plates

Equipment used:

• Autoclave

• Plate reader

• Shaker in autoclave room

• Bunsen burner

• 37°C incubator in room 301

Protocol: iGEM protocols used for LB, LB+agar. Interlab protocols used for interlab OD and CFU measurements.

Observations:

Next Steps:

• Count colonies on CFU cell measurement plates for interlab cell measurement protocol

• Transform pET28a+arg1intoMG1655ΔlacI cells

Day 5, Friday 15/06

People In Lab:Ahmed, Amalia, Nina, Tashi

Agenda:

• Check overnight plated cultures for interlab CFU count

• Transformation of pET28a+arg1 into electrically competent MG1655ΔlacIcells

Observations:

Interlab CFU count

Miniscule colonies on some plates after ~11 hours of incubation at 37°C. Some plates did not have any colonies. Plates were put back in the incubator for a few more hours.

Equipment used:

• 37°C incubator in room 301

• Shaker in room 315

• Electroporator

Protocol: iGEM protocols used for transformation by electroporation.

Transformation of ARG1 in electrically competent MG1655ΔlacIcells

• pET28a+arg1diluted from 39.3ng/mL to 1ng/mL

  • 38.3μLnuclease free water + 1μLpET28a+arg1

• transformed cells in cuvette placed in shaker at 37°C, 250rpm

• 3 plates

  • 40μLof untransformed MG1655ΔlacIcells (negative control)

  • 100μLof MG1655ΔlacItransformed with pET28a+arg1

  • 200μLof MG1655ΔlacItransformed with pET28a+arg1

Next Steps:

• Redo Calibration #2: Particle Standard Curve - Microsphere Protocol

• Overnight culture of 2B (2 colonies) and 2D (2 colonies) in DH5α

• If pET28a+arg1 transformation works

  • 2 MG1655ΔlacI overnight cultures (LB+KAN+IPTG)

  • 2 MG1655ΔlacI overnight cultures (LB+KAN)

  • streak a single MG1655ΔlacIcolony on an LB+KAN plate

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