Difference between revisions of "Team:UESTC-China/interlab"

Background

In order to repeat measurements in different labs, iGEM has proposed Interlab studies. The aim is to improve the measurement tools available to both the iGEM community and the synthetic biology community as a whole. This is an optional and voluntary exercise for all teams. This is also an optional activity for one the <a href="https://2017.igem.org/Judging/Medals">Bronze Medal Requirements</a> this year. This year’s gaol was to measure the GFP fluorescence in a plate reader to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable.

Overview

Reliable and repeatable measurement is a key component to all engineering disciplines. So one of the big challenges in synthetic biology measurement is that fluorescence data usually cannot be compared because it is reported in different units or because different groups process data in different ways. Often working around this by doing some sort of “relative expression” comparison; however, being unable to directly compare measurements makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.

Thanks to iGEM’s interLab protocol for measuring GFP fluorescence, we have a chance to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. Our team also participated in the measurement study <a href="https://2016.igem.org/Team:UESTC-China">last year.</a> Through learning from them, we have confident in doing this better this year. With minor difficulties we managed to complete the experiments and submit our results.

Devices

The 2017 iGEM teams were provided with six test devices, including two controls, a positive and negative control.

<a href="http://parts.igem.org/assembly/plates.cgi?id=4995">Kit Plate 7 InterLab Part</a> Locations.

<img src="" style="width: 80%;" />

Each of the test devices consisted of promotors J23101, J23106, J23117, J23101, J23106, J23117 respectively. The rest of the test devices was identical consisting of the RBS, the terminator and the GFP (E0040).

Each device is in the pSB1C3 plasmid backbone, so transform all of the devices onto plates containing chloramphenicol.

Materials and Methods

Thermo MULTISKAN GO plate reader

HITACHI F-4600 Fluorescence spectrophotometer

Competent cells (Escherichia coli strain DH5α)

We do our interlab study depends on <a href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">Plate Reader</a> Protocol

Results and Discussion

<img src="" style="width: 55%;" /> <img src="" />

Fig.1 Gel Electrophoresis all devices including positive control and negative control.

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Fig.2 FITC fluorescence standard curve. This is the result of serial two-fold dilutions of FITC from the stock solution of 50μM. The value of R-squared is 0.9762.

<img src="" style="width: 60%;" />

Fig.3 Fluorescence over 6 hours for partial devices.

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Fig.4 Fluorescence over 6 hours for the remaining devices.

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

Conclusion

In Fig.2 and Fig.3, we compare the same promoter in different devices and at the same time, we reserve the positive control and negative control in both of the Figs.

We draw the conclusion that Device 2 has stronger promoter than Device 1and 3, and Device 5 has stronger promoter than Device 4and 6. Moreover, Device 1, 2 and 3 has stronger promoter than Device 4, 5 and 6 on average although they all have similar variation trend of OD600. (We don’t give this Fig )

However in the Fig.5, we can’t get any obvious result even though the devices we get have the target bands in Fig.1. So we think the instruments are very import because we use a Fluorescence spectrophotometer rather than a Plant Reader. But we can promise both the fluorescence and OD600 of each device are accurate and have ideal trend.

Notebook

Week 1:

• 1. OD600 Reference point
• We ues Thermo MULTISKAN GO plate reader to complete this part of measurement. And we record the best results in excel from several data.
• 2. Protocol fluorescein fluorescence standard curve (Prepare the fluorescein stock solution, Prepare the serial dilutions of fluorescein)
• As we lack a Plate Reader to get fluorescence, so we have to use our only HITACHI F-4600 Fluorescence spectrophotometer to get results. So in this part, we can predict the results may be as ideal as others’.

Week 2:

• 1. Transform the InterLab Parts
• Device 1 is more difficult to transform than other parts, we cost 4 days to achieve it.
• 2. Cell measurement protocol
• We also use HITACHI F-4600 Fluorescence spectrophotometer to measure fluorescence.

Reference

Beal, J., Haddock-Angelli, T., Gershater, M., de Mora, K., Lizarazo, M., Hollenhorst, J., & Rettberg, R. (2016). Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PloS one, 11(3), e0150182.