Team:LZU-China/interlab

       In our interlab work we test the three required parts. They are:

•               1. BBa_I20260 (J23101-B0032-E0040-B0015) Plate 4,Well 18A

•               2. BBa_J23101 + BBa_E0240 (B0032-E0040-B0015) Plate 1,Well 20K and Plate 2,Well 24B 

           3.BBa_J23115 + BBa_E0240 (B0032-E0040-B0015) Plate 1,Well 22I and Plate 2,Well 24B

                                          Figure-1 The gel picture of each device

                                                                   Table-1 Each lane’s properties

Equipment and chemicals

   20μL eppendorf tubes x9

   ddH2O

   Competent E.Coli

   The devices

   Fluorescence microscope.

Methods:

   Each device was transformed in competent E.Coli and shaken cultured at 37℃ for 18h. Then we detected the number of green light spot by software. The final result is the average light spot , which are more bright than G value 192 for 3 random sites of each site by the algorithm which we used to developed.

            Picture 1-3 are the device 1's results, Picture 4-6 are the device 2's and the Picture 7-9 are the device 3's. Each picture is 400x and we calculated the light spot. The result are listed here:

                                                 Figure-3 The "light" spot number of each devices

                                                  Table-3 The mean and standard deviation of each device

                     Besides, we used the cytoflower to detect the number of cells with green fluorescence. The result is here: (Tube 001 is the control. Tube 002 and 003 are Device 2 . Tube 004 is Device 1 and Tube 005 is Device 3. )

1.Who did the actual work to acquire these measurements?

Qi Wu, Hongxia Zhao and Ruixue Zhao.

2.What other people should be credited for these measurements? (i.e., who would be an author on any resulting publication. For example, your faculty advisor may have helped design the protocols that you ran.)

Xiangkai Li will be the "corresponding author".

3.On what dates were the protocols run and the measurements taken? (this will often be a range of dates; make sure you say which data was taken at what times.)

We ran the protocol on2014.10.4 because of the mistake and we took the simple fluorescence data on 2014.10.16. The data made by flow cytometer was got on 2014.10.17.

4.Do all persons involved consent to the inclusion of this data in publications derived from the iGEM interlab study?

Yes.

1.What protocol did you use to prepare samples for measurement?

We used the Biobrick protocol to assembly and the strain was DH5 Alpha.

2.What sort of instrument did you use to acquire measurements?

o What is the model and manufacturer?

Fluorescence microscope is Nikon OPTIPHOT-2 .

o How is it configured for your measurements? (e.g., light filters, illumination, amplification)

The microscope's exciting light was set as 492nm.

3.What protocol did you use to take measurements?

The transformed E coli. Was shaken cultured about 17h and we took photo by the fluorescence microscope.Then we analyzed the number of light spots by software.

4.What method is used to determine whether to include or exclude each sample from the data set?

After the scoping we did electrophoresis for each tube to exam if the bacteria had been transformed.

5.What exactly were the controls that you used?

DH5 Alpha without transforming which had been shaken cultured for 17h.

6.What quantities were measured? (e.g., red fluorescence, green fluorescence, optical density)

Green fluorescence. For the three reporters are all GFP.

7.How much time did it take to acquire each set of measurements?

The data was got on the same day. While different device was transformed successfully on different date. Device 1 took more time, about 7 days and was got on October 13th. Device 2 and 3 were transformed successfully on 10.14, only taking 3 days.

8.How much does it cost to acquire a set of measurements?

We did not calculate this carefully and we can just give a estimated value. We cost about 1300RMB for this measurement, 800 for the reagent and 500 for the use of instrument.

9.What are the practical limits on the number or rate of measurements taken with this instrument and protocol?

pSB3K3 has a lower copy number so we had to cultured it for more times to select the colony of Device 1.

1.For each type of quantity measured (e.g., fluorescence, optical density), report on the following:

2.Units:

o What are the units of the measurement? (e.g., meters, molecules)

The number of light spot.

o What is the equivalent unit expressed as a combination of the seven SI base units? (http://en.wikipedia.org/wiki/SI_base_unit)

We just calculated the number, it does not need the SI unit.

3.Precision:

o What is the range of possible measured values for this quantity, using your instrument as configured for these measurements? (e.g., a meter stick measures in the range of 0 to 1 meter)

The spot number is always rational number while the RGB G value range is 0-255.

o What are the significant figures for these measurement? (e.g., on a meter stick, it is common to measure to the nearest millimeter).

Both the number and G value has the precision of 1.

o Is the precision the same across the entire range? If not, how does it differ?

Yes.

o How did you determine these answers?

Our method determined that we couldn't get a discrete result like 0.002. The method is counting instead of detect.

4.Accuracy:

o When was the instrument last calibrated?

2014.8.11.

o How was the instrument calibrated?

Calibrating the light source by raster correction

1.For each sample, report:

• o the identity of the sample

 1-3:Device1; 4-6:Device2; 7-9:Device3.

• o each quantity directly measured

They are saved as picture in the .rar file.

• o each quantity derived from measurements (e.g., fluorescence/OD)

2.For each group of replicates, report:

• o the identity of samples in the set

           1-3:Device1; 4-6:Device2; 7-9:Device3.

• o which, if any, of the samples are excluded and why

  No excluded sample.

• o the mean and standard deviation for each quantity measured or derived