Team:HZAU-China/InterLab

Mixed-Reality CellBidirectional coupling between real and virtual bio-oscillators



Overview


This year, we are not only in general event but also participate in Measurement Interlab. All the iGEM teams that are in this Interlab Study should collect fluorescence intensity data of three specific genetic devices which is the same with others and can express GFP protein.


The three genetic devices differ in their promotors :

Device 1: J23101 + I13504 (B0034-E0040-B0015), in the pSB1C3 backbone

Device 2: J23106 + I13504 (B0034-E0040-B0015), in the pSB1C3 backbone

Device 3: J23117 + I13504 (B0034-E0040-B0015), in the pSB1C3 backbone


Firstly, we transformed these three devices into competent cells (DH 5 alpha) respectively. And then, We did pre-experiment before formal experiment to confirm the suitable period as well as the dilution factor. What’s more, their fluorescence intensity is measured with a plate reader in suitable optical density of the LB medium. We assumed that the GFP expression quality of them ordered from high to low would be Device 1, Device 2, Device 3. Finally, the result confirmed our conjecture.





Protocol


Assembly

All constructs were taken directly from the iGEM 2015 distribution plates. We used biobrick assembly.


Click here to get Plasmid profiles of three devices.

Plasmid1 profile of three devices.

Plasmid2 profile of three devices.

Plasmid3 profile of three devices.


The correct identity of the resulting constructs were confirmed by sequencing.Click here get nucleotide sequences.


Transforming these three devices into competent cells (DH 5 alpha) respectively. Incubate E.coli with 20 ml LB medium at 37℃ and 180 rpm. Add appropriate antibiotics samples (chloramphenicol for J23101, J23106, J23117 and all devices. Ampicillin for I13504.). Both antibiotics were added from 1000x stock stored at -20℃ for a final concentration of 50 μg/ml for Ampicillin and 20 μg/m for chloramphenicol, respectively.


Per-experiment

Here is some reason we did pre-experiment.

(1) Confirm the best dilution factor.

(2) Confirm the suitable period for measurement.

(3) Draw a growth curves.


Formal experiment

1. We set 3 groups except 3 experimental groups (Device 1, Device 2 and Device 3).One of these groups was negative control (GFP at PSB1C3.) and the positive control is R0040,which is pTetR in pSB1C3.Last we used LB medium as background. All groups were triplicate.


2. We incubated samples in 5 ml centrifuge tubes, then measuring at 96 wells plates. We used SynergyTM H1 Multi-Mode Reader to measured fluorescence intensity and optical density. There is the website of this instrument:

http://www.biotek.com/products/product_print.html?newsid=10711

Because there is a OD detector on the top of 96-well plate, pathlength of light would affect the optical density.

A=ε b c.

We add LB medium into wells to dilute sample will lengthen pathlength of light.

x: the volume of media to dilute the samples.

It is clear that A’=A, diluting sample will not work.

We added more samples to lengthen pathlength but the concentration of biomass will not be changed.

x =μl.

(1) Add 200μl sample to measure optical density.

(2) Use formula to calculate the volume for adjusting the OD600.

(3) Use silver paper to pack 5ml centrifuge tubes (for shaking culture) to eliminate the influence of illumination.


3. Here is the protocol of Multi-Mode Reader.

          Type of plate 96 WELL PLATE

          Set temperature 37℃

          Excitation: 485, Emission: 511

          Optical elements: top, gain: auto

          Light source: xenon stroke light

          Detecting height: 7 mm

          Measuring optical density 600

          Wave length: 600 nm

          Optical path enlargement emendation: 977/900

          The value of 1 cm absorbancy: 0.18




Results


1. Here are results of per-experiment.

The growth curve of bacteria was s-shaped. The data of OD600 reached the top when cells were incubated for 11 hours and half of maximum for about 9 hours.


Fig.1 Growth curve of per-experiment As is shown below, the maximum of this curve is about 0.133 at 16 hours and later then it begins to drop down. It is about half of maximum when sample was incubated about 9 hours, so we choose 9 hours as measuring situation.

2. Here are results of formal experiment

Each sample here was given in arbitrary units-fluorescence intensity/OD600.

We used 5ml centrifuge tubes to culture our samples and they were measured by a plate reader after 9 hours’ incubating. Three separate colonies were taken from the same sample.


The following is our calculation method.

F=F1-F0

O=O1-O0

F/O is the final data.

F1 : Fluorescence intensity of experimental groups(include three devices) .

F0 : Fluorescence intensity of negative control(E.coli with I13504 in pSB1C3).

O1: Optical density of experimental groups (include three devices) .

O0: Optical density of background(LB media).


Fig.2 A1-A9 are the measured values of Device1 and A1-A3 are one of the measured values of technical repeats. A1-A3, A4-A6 and A7-A9 are biological repeats respectively.B1-B9 and C1-C9 are the measured values of Device 2 and Device 3 respectively. D1-D9 are the measured values of positive control while E1-E9 are the measured values of negative control. F1-F9 are values of background(LB medium).

Fig.3 Device 1: J23101 + I13504 (B0034-E0040-B0015), in the pSB1C3 backbone. Device 2: J23106 + I13504 (B0034-E0040-B0015), in the pSB1C3 backbone. Device 3: J23117 + I13504 (B0034-E0040-B0015), in the pSB1C3 backbone.


© 2015 Huazhong Agricultural University iGEM Team. All rights reserved.

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