Team:Gifu/InterLab

 

INTERLAB





[Introduction]
We joined InterLab Measurement Study in this year.The objective of this year’s iGEM Interlab study is to quantify expression of five different reporter constructs.

[Materials and Methods]
Materials
・Safire ART-NR:F129013
It is a plate reader to measure absorbance and fluorescence of samples.

Methods
1. Calculate the OD600 Reference point
Measure absorbance 600 nm of LUDOX-S30 and H2O in a 96 well plate in our plate reader. It is to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement.

2. Generate the FITC fluorescence standard curve
Prepare a dilution series of FITC in 4 replicates and measure the fluorescence in a 96 well plate in our plate reader. By measuring these in all standard modes, we can generate a standard curve of fluorescence for FITC concentration.

3. Measure cell
Day1 : Transform Escherichia coli these plasmids.
Plasmids
・Positive control
・Negative control
・Device1 : J23101 + I13504
・Device2 : J23106 + I13504
(We couldn’t transform E. coli with device3.)

Day2 : Pick 2 colonies from each of plate and glow the cells in LB medium + Chloramphenicol overnight. (37C and 220ppm)

Day3 : Cell growth, sampling, and assay
Measure OD600 of these cells cultures and dilute it to a target OD600 of 0.02 in 10 mL 0.5×TB medium + Chloramphenicol and incubate the cultures (37C and 220rpm).At 0,1,2,3,4,5,and 6 hours of incubation take 100 μL samples of the cultures. At the end of sampling point, measure Absorbance 600 nm and Fluorescence of these samples. (Use same instrument setting when measuring the FITC standard curve.)

[Result]
1. Calculate the OD600 Reference point
The Abs600 data of LUDOX and H2O is Table1.
And correction factor to transform our absorbance data into a standard OD600 measurement is calculated by the following formula.
Correction factor = Reference OD600 / (LUDOX average – H2O average)

   Table 1. Absorbance 600 nm of LUDOX-S30 and H2O

LUDOXH2O
replicate 10.04850.0387
replicate 20.04820.0395
replicate 30.05790.0377
replicate 40.05890.0384
Average0.05340.0386
Corrected Abs6000.0148---
Reference OD6000.0148---
Correction factor0.0997---


2. Generate the FITC fluorescence standard curve
The fluorescence data of FITC is table2. And FITC standard curve is figure1. We couldn’t measure the data of FITC concentration from 250 μM to 15.625 μM. Because we failed the settings.

   Table 2. fluorescence data of FITC in any dilution

Concentration (μM)7.813.911.950.9770.4880.2440
replicate 14021120994145257764685849584502
replicate 24694424730152969009783562345512
replicate 34638426163142719337785846133915
replicate 44542724076126328586565842732712
Average4474223991141818674705250204160
S.D.30852180112168010408571169




Fig.1


3. Measure cell 

  Table 3.

SampleAbs600 readingVolume of preloading cultureVolume of preloading media
Positive control0.07999.260.741
Negative control 0.069018.7-8.69
Device10.08148.661.34
Device20.08059.010.991
Media+chl0.0583------



   Table 4. blank substraction and correction (bacteria concentration)

Positive control 1Positive control 2Negative control 1Negative control 2Device 1-1Device 1-2Device 2-1Device 2-2
0 h0.020400.018610.095390.013920.021900.0210.0096390.01113
1 h0.041030.039340.025680.030870.028890.027580.027580.02897
2 h0.10350.10140.095950.10290.055980.53490.098340.09804
3 h0.18320.18410.16250.17890.10730.10630.16080.1702
4 h0.32870.33130.31760.33940.18750.18100.30760.3090
5 h0.49610.50370.47220.52530.33890.33100.48580.4858
6 h0.71030.71910.69080.74180.55240.54560.71160.7067



Fig.2


  Table 5. blank substraction (fluorescence)

nullPositive control 1Positive control 2Negative control 1Negative control 2Device 1-1Device 1-2Device 2-1Device 2-2
0 h455053761488941182151851939564360
1 h61666942414294180641836236884091
2 h1842819780-426-255269462759768007414
3 h2561927051-495-34033766350921023310839
4 h3225834499-652-48050905519131470415534
5 h4761750169-802-494-8413-84132503225931
6 h-8413-8413-693-439-8413-84133588735386



Fig.3


  Table 6. Value of Flu/Abs600

nullPositive control 1Positive control 2Negative control 1Negative control 2Device 1-1Device 1-2Device 2-1Device 2-2
0 h22302528891415601267595831851881857410449391624
1 h150277176476161279532625632665830133738141207
2 h178015195018-4438-24764813405159316915175625
3 h139881146900-3044-19003146673300906362963687
4 h98152104118-2052-14142714432868804779950269
5 h9598599608-1698-940-24822-254135152553376
6 h-11845-11699-1003-592-15230-154195043550074


  Table 7. Average and S.D.

AveragePositive controlNegative controlDevice 1Device 2S.D.Positive controlNegative controlDevice 1Device 2
0 h25597011180348568544010360 h46591625203536013311
1 h163376128296457311374721 h185254664284245281
2 h186517-3457498636723882 h120231388244604578
3 h143390-2472322379636583 h49638091090641
4 h101135-1733279161490344 h4219452109161747
5 h97797-1319-25117524505 h25625364171308
6 h-11772-797-15324502546 h103291134255




Fig4.