Team:HUST-China/InterLab

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Interlab

Introduction

It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by measure the expression level of GFP, in order to help the iGEM community collect data about how reliable will these devices turn out to be in labs around the world.

Provenance and release

What chassis did you use?

Escherichia coli DH5α

What Biosafety Level is your chassis?

BSL1

What PPE did you utilize during your experiments?

T ianming gloves Songxinjiujiu labcoats

Instrument

What instrument did you use during your measurements?

plate reader

Please provide the brand and model of your instrument.

Flexstation 3

Calibration protocol

A1. OD600 reference point-LUDOX protocol

Did you use pathlength correction during measurement? Yes

Number of flashes per well

6

Orbital averaging (nm)

600

What temperature setting did you use during the measurement? 22℃

What type of 96-well plate did you use?

Black plate (preferred)

Did your plate have flat-bottomed or round-bottomed wells?

Flat

Did you use pathlength correction during measurement?

Yes Number of flashes per well 6

Orbital averaging (nm)

600 What temperature setting did you use during the measurement? 22℃ What type of 96-well plate did you use? Black plate (preferred)

Did your plate have flat-bottomed or round-bottomed wells?

Flat

B2. Measurement Steps

A2. Measurement Steps（more details）

Add 100ul LUDOX into wells A1, B1, C1,D1 Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements

Add 100ul ddH2O into wells A2, B2, C2,D2 Record the data in the table below or in your notebook Import data into Excel sheet provided (OD600 reference point tab) B1. Particle standard curve-microsphere protocol

Prepare the Microsphere Stock Solution: （more details）

Add 100ul LUDOX into wells A1, B1, C1,D1

Add 100ul ddH2O into wells A2, B2, C2,D2

❏ Obtain the tube labeled “Silica Beads” from the InterLab test kit and vortex

❏vigorously for 30 seconds. NOTE: Microspheres should NOT bestored at 0°C or below, as freezing affects the properties of the microspheres. If you believe your microspheres may have been frozen, please contact the iGEM Measurement

Committee for a replacement (measurement at igem dot org).

❏ Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube

❏ Add 904 μL of ddH2O to the microspheres

❏ Vortex well. This is your Microsphere Stock Solution.

Prepare the serial dilution of Microspheres: （more details）

Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN ddH2O ONLY.

Initially you will setup the plate with the microsphere stock solution in column 1 and an equal volume of 1x ddH2O in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.

❏Add 100 μl of ddH2O into wells A2, B2, C2, D2....A12, B12, C12, D12

❏Vortex the tube containing the stock solution of microspheres vigorously for 10 seconds

❏Immediately add 200 μl of microspheres stock solution into A1

❏Transfer 100 μl of microsphere stock solution from A1 into A2.

❏Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…

❏Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...

❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...

❏Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...

❏Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...

❏Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...

❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...

❏Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...

❏Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...

❏Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste

TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.

❏ Repeat dilution series for rows B, C, D

❏ IMPORTANT! Re-Mix (Pipette up and down) each row of your plate immediately

before putting in the plate reader! (This is important because the beads begin to settle to the bottom of the wells within about 10 minutes, which will affect the measurements.) Take care to mix gently and avoid creating bubbles on the surface of the liquid.

❏ Measure Abs600 of all samples in instrument

❏ Record the data in your notebook

❏ Import data into Excel sheet provided (particle standard curve tab)

C1: Fluorescence standard curve - Fluorescein Protocol

Did you use pathlength correction during measurement?

Yes

Number of flashes per well

6

What gain setting did you use?

Automatic

If you used a filter, what light wavelengths did it pass?

530nm

Emission wavelength

525nm

Excitation wavelength

485nm

Fluorescence reading

Bottom optic

What type of 96-well plate did you use?

Black plate (preferred)

Did your plate have flat-bottomed or round-bottomed wells?

Flat

What temperature setting did you use during the measurement?

22℃

C2. Measurement Steps

Prepare the fluorescein stock solution: （more details）

❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. [ Note: it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up anddown and examine the solution in the pipette tip – if anyparticulates are visible in the pipette tip continue to mix the solution until they disappear.]

❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1x PBS

Prepare the serial dilutions of fluorescein: （more details）

❏Accurate pipetting is essential. Serial dilutions will be performed across columns 1-11. COLUMN 12 MUST CONTAIN PBS BUFFER ONLY.

❏ Initially you will setup the plate with the fluorescein stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. You will perform a serial dilution by consecutively transferring 100 μl from column to column with good mixing.

❏Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12

❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1

❏ Transfer 100 μl of fluorescein stock solution from A1 into A2.

❏ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…

❏ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...

❏Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...

❏ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...

❏ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...

❏ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...

❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...

❏ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...

❏ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...

❏ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.

❏ Repeat dilution series for rows B, C, D

❏ Measure fluorescence of all samples in instrument

❏ Record the data in your notebook

❏ Import data into Excel sheet provided (fluorescein standard curve tab)

Cell culture setup and meaturement

Transformation:

Negative control BBa_R0040 Kit Plate 7 Well 2D

Positive control BBa_I20270 Kit Plate 7 Well 2B

Test Device 1 BBa_J364000 Kit Plate 7 Well 2F

Test Device 2 BBa_J364001 Kit Plate 7 Well 2H

Test Device 3 BBa_J364002 Kit Plate 7 Well 2J

Test Device 4 BBa_J364007 Kit Plate 7 Well 2L

Test Device 5 BBa_J364008 Kit Plate 7 Well 2N

Test Device 6 BBa_J364009 Kit Plate 7 Well 2P

Cell grow:: ❏Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.

Cell growth, sampling, and assay

❏Make a 1:10 dilution of each overnight culture in LB+Chloramphenicol (0.5mL of culture into 4.5mL of LB+Chlor)
❏Measure Abs600 of these 1:10 diluted cultures
❏Record the data in your notebook
❏ Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 ml LBmedium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light).
❏ Take 500 µL samples of the diluted cultures at 0 hours into 1.5 ml eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice.

❏ Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours.
❏ Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 ml eppendorf tubes. Place samples on ice.
❏ At the end of sampling point you need to measure your samples (Abs600 andfluorescence measurement), see the below for details.
❏ Record data in your notebook.
❏ Import data into Excel sheet provided (fluorescence measurement tab)