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Week 1 Week 2 Week 3

Week 1

Day 1, Monday 04/06

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

Agenda:

• LB media

• LB + agar plates

• Aliquoted autoclaved SOC

• 70% EtOH

Equipment used:

• Autoclave

• Bunsen burner

Protocol: iGEM protocols used for LB, LB+agar, SOC (altered to accommodate MgCl2·6H₂O because we didn't have anhydrous version, thus, for 1L solution, 2.17g was needed).

Next Steps:

• Get MG1655ΔlacI cells from Christian and streak onto LB plate

• Transform arg1 and pSB1C3+RFP (iGEM competence test plasmid) into DH10β

• Make MG1655ΔlacI glycerol stocks

• Make LB+CAM and LB+KAN plates

Day 2, Tuesday 05/06

People In Lab: Carla

Agenda:

• KAN plates

• CAM plates

Equipment used:

• Autoclave

• Bunsen burner

Protocol: iGEM protocol used for LB+agar.

Calculation for antibiotics to be added to LB media for plating

Chloramphenicol

C₁V₁= C₂V₂

C₁= stock concentration = 25mg/mL

C₂ = working concentration = 25μg/mL

(25000μg/mL)(V₁) = (25μg/mL)(500mL)

V₁= 0.5mL

Kanamycin

C₁V₁= C₂V₂

C₁= stock concentration = 10mg/mL

C₂ = working concentration = 50μg/mL

(10000μg/mL)(V₁) = (50μg/mL)(500mL)
V₁= 2.5mL

Next Steps:

Obtain overnight culture of MG1655ΔlacI in the morning left for us in the shaker in room 318

Day 3, Wednesday 06/06

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

Agenda:

• Transform pET28a+arg1 and pSB1C3+RFP (iGEM competence test plasmid) into DH10β

• Streak MG1655ΔlacI onto LB plate

Equipment used:

• ThermoMixer

• Bunsen Burner

• 37°C incubator in room 301

Protocol: iGEM protocols used for transformation.

Transformation

Transformation 1: pSB1C3+RFP(10pg/μL) in DH10β: 100μL plated on LB+CAM

Transformation 2: pSB1C3+RFP (10pg/μL)in DH10β: 100μL plated on LB+CAM

Transformation 3: pET28a+arg1 in DH10β: 100μL plated on LB+KAN

Transformation 4: pET28a+arg1 in DH10β: 100μL plated on LB+CAM

Negative control: 100μL DH10β plated on LB+CAM and LB+KAN

For each transformation, 50μL of cells and 2μL of plasmid, and 250μL of SOC were used

Next Steps:

Start interlab

Day 4, Thursday 07/06

People In Lab: Ahmed, Amalia, Daniel, Jasmeen, Nina

Agenda:

• Observed transformed cells

  • pSB1C3+RFP (10pg/μL) in DH10β

  • pET28a+arg1 in DH10β

• Observe streaked plate (LB+MG1655ΔlacI)

• Make overnight cultures from transformed cells (37°C)

  • 3 LB+CAM+DH10β+pSB1C3+RFP

  • 3 LB+KAN+DH10β+pET28a+arg1

  • 1 LB (negative control)

• Transform interlab plasmids into DH5α

• 10 nuclease-free water aliquots

Equipment used:

• Thermomixer

• Bunsen Burner

• 37°C incubator in room 301

Protocol:Interlab protocols used for interlab transformation.

Interlab transformation

• resuspend plasmids from plate 7 to a concentration of 200-300pg/μL

• 1μl plasmid used: 2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P

• 50μL of competent cells used (DH5α)

• 50μL of cells plated on LB+CAM

• 2J was not incubated, has to be transformed again

Observations:

Picture of plates are shown below.

100μl transformation pET28a+arg1 in DH10β on LB+KAN

100μl transformation pSB1C3+RFP in DH10β on LB+KAN

100μl negative control (DH10β no plasmid) on LB+KAN and LB+CAM

Streaked MG1655ΔlacI on LB

Next Steps:

• Miniprep pSB1C3+RFP and pET28a+arg1 from overnight cultures

• Interlab calibration #1: OD₆₀₀ Reference point - LUDOX Protocol

Day 5, Friday 08/06

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

Agenda:

• Observe interlab transformations

• Miniprep pSB1C3+RFP and pET28a+arg1 from overnight cultures

• Nanodrop

• Send miniprepped pSB1C3+RFP and pET28a+arg1 to Ranomics to add UNS2 and UNS3 to pSB1C3+RFP and fix illegal cut sites in pET28a+arg1

• Interlab calibration #1: OD₆₀₀ Reference point - LUDOX Protocol

Equipment used:

• Thermomixer

• Bunsen burner

• 37°C Incubator in room 301

• Centrifuge

• Nanodrop

• Plate reader

Protocol: Protocol from miniprep kit used for miniprep. Interlab protocol used for Interlab calibration #1: OD₆₀₀ Reference point - LUDOX Protocol.

Miniprep of pSB1C3+RFP and pET28a+arg1

• 10mL of each overnight culture used

• 60μL of nuclease-free water used for each elution

Sending miniprepped pSB1C3+RFP and pET28a+arg1 to Ranomics

(Concentration after nanodrop)(Volume sent) = (mass sent)

pSB1C3+RFP: (39.3ng/μL)(51μL) = 2004.3ng

pET28a+arg1: (142.0ng/μL)(21μL) = 2982ng

Observations:

Interlab Transformation of 2F, 2H, 2N on LB+CAM

Interlab Transformation of 2B, 2D, 2L, 2P on LB+CAM

Interlab calibration #1: OD₆₀₀ Reference point - LUDOX Protocol absorbance values

*Refer to interlab page on wiki*

Next Steps:

• Transform 2J plasmid into DH5α

• Make LB+KAN+CAM plates

• Make PBS (dilute from stock in fridge or make from scratch)

• Transform pET28a+arg1and pSB1C3+RFP into MG1655ΔlacI

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

Week 2

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

Week 3 NOT DONE

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