Team:SSTi-SZGD/InterLab

Overview

We are very delighted to take part in this year's International interlaboratory measurement study

We finished the following five tasks this year:

  1. OD600 Reference point - LUDOX Protocol
  2. Particle Standard Curve - Micro-sphere Protocol
  3. Fluorescence standard curve - Fluoresce in Protocol
  4. Cell measurement protocol
  5. Colony Forming Units per 0.1 OD600 E. coli cultures

The six devices were tested this year are shown as follows, along with the controls:

  • Positive control: BBa_I20270
  • Negative control: BBa_R0040
  • Test Device 1: BBa_J364000
  • Test Device 2: BBa_J364001
  • Test Device 3: BBa_J364002
  • Test Device 4: BBa_J364007
  • Test Device 5: BBa_J364008
  • Test Device 6: BBa_J364009
Protocol

Calibration 1: OD600 Reference point - LUDOX Protocol

  • Add 100μl LUDOX into wells A1, B1, C1, D1
  • Add 100μl of ddH 2 O into wells A2, B2, C2, D2
  • Measure absorbance at 600nm of all samples in the measurement mode you plan to use for cell measurements

Calibration 2: Particle Standard Curve - Micro-sphere Protocol

Pipet 96μl microspheres into a 1.5ml Eppendorf tube, add 904μl of ddH 2 O to the micro-spheres and vortex well. Add 100μl of ddH 2 O into wells A2, B2, C2, D2…A12, B12, C12, D12 ; vortex the tube vigorously for 10s then immediately add 200μl of micro-spheres stock solution into A1; A serial dilution was performed by consecutively transferring 100ul from column to column with good mixing.
Then repeated serial dilution for rows B,C and D. Measured fluorescence in standard measurement modes on plate reader, then imported data into Excel.

Calibration 3: Fluorescence standard curve-fluoresce in Protocol

Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. Prepare 10×fluorescein stock solution(100uM) by resuspending fluorescein in 1ml of 1×PBS.
Dilute the 10×fluorescein stock solution with 1×PBS to make a 1×fluorescein solution and the resulted concentration of fluorescein stock solution was 10uM. The plate was set up with the fluorescein stock in column 1 and an equal volume of 1×PBS in columns 2 to 12. A serial dilution was performed by consecutively transferring 100ul from column to column with good mixing.
Then repeated serial dilution for rows B, C, and D. Measured fluorescence in standard measurement modes on plate reader, then imported data into Excel.

Cell measurement protocol

Day 1: transform Escherichia coli DH5α with these following plasmids (all in pSB1C3) BBa_I20270 BBa_R0040 BBa_J364000 BBa_J364001 BBa_J364002 BBa_J364007 BBa_J364008 BBa_J3640098
Day 2: Pick 2 colonies from each of the transformation plates and inoculate in 5-10ml LB medium Chloramphenicol. Grow the cells overnight (16-18hours) at 37℃ and 220rpm.
Day 3: Cell growth, sampling, and assay. In total two plates were set up to read, one plate was for each time point respectively: 0 and 6 hours. Each plate was set up as shown below. Measured absorbance at 600nm and measured fluorescence in standard measurement modes on plate reader, then imported data into Excel.

Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

Step 1: Starting Sample Preparation
Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of your plate reader to 0.05–0.5 OD600 range. Check the OD600 and make sure it is 0.1(minus the blank measurement).

Step 2: Dilution Series Instructions
Make 1:20 dilutions for dilutions 1, 2 and 3 using 2.0ml tubes with 1900µl of LB + Cam media. Make 1:10 dilutions for dilutions 4 and 5 using 1.5ml tubes with 900µl of LB + Cam media. Aseptically spread plate 100µl on LB + Cam plates for Dilutions 3, 4, and 5 and finally incubate at 37℃ overnight and count colonies after 18-20hours of growth.

Step 3: CFU/mL/OD Calculation Instructions
Count the colonies on each plate with fewer than 300 colonies.
Colonies × Final Dilution Factor=CFU/mL

LUDOX CL-X H2O
Replicate 1 0.056 0.035
Replicate 2 0.056 0.036
Replicate 3 0.056 0.037
Replicate 4 0.055 0.035
Arith. Mean 0.056 0.036
Corrected Abs600 0.020
Reference OD600 0.063
OD600/Abs600 3.150

Conclusion

This year’s interlab tested the results of combination of different promotors with the same RBS sequence. According to the experiment: 1) Device 1 (J23101+B0034) increased GFP protein’s expression but hindered the growth of bacteria; 2) Device 2 (J23106+B0034) The GFP protein wasn't expressed in a large quantity, but the bacteria grew well compared to other devices; 3) Device 3 (J23117+B0034) The bacteria grew normal but the GFP protein expression wasn’t comparably high ; 4) Device 4 (J23100+B0034) GFP protein expression was the highest and the bacteria grew normal; 5) Device 5 (J23104+B0034) The bacteria failed to grow well and the expression level of GFP protein was low; 6) Device 6 (J23116+B0034) The bacteria grew well but the GFP protein’s expression level was somehow very low. In conclusion, device 4 (J23100+B0034) is the best and device 3 (J23117+B0034) has a low efficiency.

Copyright © 2018 SSTi-SZGD All Rights Reserved.
Copyright © 2018 SSTi-SZGD All Rights Reserved.