Team:CGU Taiwan/Interlab

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Abstract
Protocols
Results
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Notebook





Interlab



Abstract

We often used fluorescence as an indicator of promoter activity. However, it is difficult to compare fluorescence measured from different laboratories because the data are reported in difference units or shown in a different way. To solve this problem, iGEM Interlab study tries to transform fluorescence from a relative unit into an absolute unit and then can be used over the world.

This year's iGEM Interlab study is to quantify expression of five different plasmids and compare the results from various teams. iGEM Interlab study has provided 2 protocols of measuring GFP, and we choose “plate reader”. We have to measure absorbance at 600 nm for GFP expression per cell for all plasmids (or devices).

These 5 plasmids have GFP under different promoters and ribosome binding sequences (RBS) in the pSB1C3 plasmid backbone. To verify if the plasmids are correct, we calculated the DNA fragment sizes of all device. They are shown below:





Protocols

We followed the protocol of iGEM InterLab measurement study to measure absorbance at 600 nm of LUDOX and made a FITC fluorescence standard curve.

As the very first step of the cell measurement experiment, we began by verifying the accuracy of plasmids given by iGEM Interlab study: Device 1, Device 2, Device 3, Positive Control, Negative Control. First, we transformed the plasmids into competent bacteria DH5α. Plasmids were purified from cultures of the positive colonies. We then performed PCR to confirm that all plasmids are correct. Next, we followed the plate reader protocol posted on iGEM InterLab measurement study, that can be found here. We tried many different setting for measuring fluorescence: different excitation and emission wavelengths, reading from the top or bottom of plate, and using filter or not. In the result section, we only show excitation 485nm and emission 538nm, reading from the bottom of plate, and using filter to cutoff 530nm.

You can see all the protocol here.



Materials and Instrument
Materials:

  1. Device 1: J23101.B0034.E0040.B0015 in pSB1C3
  2. Device 2: J23106.B0034.E0040.B0015 in pSB1C3
  3. Device 3: J23117.B0034.E0040.B0015 in pSB1C3
  4. Positive Control: I20270 in pSB1C3.
  5. Negative Control: R0040 in pSB1C3


Instrument:

  1. SpectraMax® M2 Multimode Plate Reader



Results


  

Fig. 1 PCR results from all devices. The PCR product of Device 1, Device 2, Device 3 and Positive Control are almost 1kb, Negative Control is at the bottom of agarose gel.




Fig.2 FITC fluorescence standard curve. This is the result of serial two-fold dilutions of FITC from the stock solution of 250μM. The value of R- squared is 0.9954.




   Fig. 3 Absorbance measured at 600nm over 6 hours for all devices.




   Fig. 4 Fluorescence over 6 hours for all devices.



   Fig. 5 The ratio of fluorescence/absorbance values measured at 600nm.



Discussion
Compare the DNA fragment size that show in abstract and figure 1, Device 1, Device 2, Device 3 and Positive Control all have 1Kb product, Negative Control has a product DNA size is less than 100bp, this matches our prediction. But the first and second sample of Device 1 (lane 2 and 3) looks different to others, we think that does not the plasmid we want. Beside that, we believe others are correct.

From figure 4, Device 1 has shown the maximum fluorescence, followed by Device 2 and Positive Control. Negative Control and Device 1 almost not detect fluorescence. Though this result, we consider Device 1 has the strongest promoter, Device 3 has the weakest promoter. In order to prove this conclusion, figure 5 shows GFP expression per cell. We realize that Device 1 is far more than others, follow by Device 2 and Device 3. This result also fit 2015 iGEM InterLab Studies (Beal et al., 2016).




Notebook



  • 8/9 and 8/10

    (1)Goal: Conduct the first experiment that is to find the OD600 reference point.

    Materials and methods:

    Transform 5 plasmids (Positive Control, Negative Control, Test Device 1, Test Device 2, and Test Device 3) from the InterLab Measurement Kit into E. coli DH5 alpha

    (2)Goal: Find the setting to produce optimal fluorescent FITC standard curve

    Pick colonies from each plates for checking the plasmids and inoculate in LB medium+Chloramphenicol. Grow the cell overnight in 37'C.

  • 8/11

    (1)Conduct the first experiment that is to find the OD600 reference point.

    Materials: Mentioned as the same as previously on 160809
    Methods: Mentioned as the same as previously on 160809

    Using Miniprep to purify plasmids, and through PCR check the correction of plasmids.

    (2)Find the setting to produce optimal fluorescent FITC standard curve.

    Materials and methods: Mentioned as the same as previously on 160810

    Start the cell measurement experiment.

  • 8/12

    Redo the cell measurement experiment.

    8/24

    Redo the cell measurement experiment.

    8/30

    (1)Find the setting to produce optimal fluorescent FITC standard curve.

    Materials:Mentioned as the same as previously on 160810

    Methods:
    1.The main change is the settings for standard GFP settings, and the rest are as mentioned as previously on 160810.
    Here are the settings of GFP settings:






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