Difference between revisions of "Team:Peking/InterLab"

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[1] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf<br/>
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[2] Woolfson, D. N., Bartlett, G. J., Burton, A. J., Heal, J. W., Niitsu, A., Thomson, A. R., & Wood, C. W. (2015). De novo protein design: how do we expand into the universe of possible protein structures?. Current opinion in structural biology, 33, 16-26.<br/>
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[2]https://static.igem.org/mediawiki/2018/e/ec/2018_InterLab_Flow_Cytometry_Protocol.pdf<br/>
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Revision as of 19:55, 16 October 2018

Interlab

Introduction

Peking 2018 joined the fifth Interlab measurement study. This year we helped to answer this question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

The introduction of this year Interlab: https://2018.igem.org/Measurement/InterLab

 

Equipment Information

Plate Reader: Perkin Elemer EnSpire TM Multilabel Reader 2300

Flow Cytometry: BD LSRFortessa TM Cell Analyzer

96 - Well Pate: Corning Incorporated Costar®️ 3603

 

Results

 

1.

Calibration 1: OD600 Reference point - LUDOX

 

Figure. 1 The result of LUDOX calibration. The correction factor of our plate reader is 3.316


 

2.

Calibration 2: Particle Standard Curve – Microsphere

 

Figure. 2 The result of particle calibration.


(a) Particle Standard Curve - Linear (b) Particle Standard Curve - Log Scale
Figure. 3 The result of particle standard curve

 

3.

Calibration 3: Fluorescence standard curve – Fluorescein

 

Figure. 4 The result of fluorescein calibration.


(a) Fluorescence Standard Curve - Linear (b) Fluorescence Standard Curve - Log Scale
Figure. 5 The result of fluorescence standard curve

 

Cell Measurement

 

Materials:

Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani) media
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light) Incubator at 37°C
1.5 ml eppendorf tubes for sample storage
Ice bucket with ice
Micropipettes and tips
96 well plate, black with clear flat bottom preferred

Figure. 6 The required test devices.



Figure. 7 The localization of each device on the 96-well plate.


1.

Plate Reader


Figure. 8 The raw readings of Abs 600 and fluorescence by the plate reader.




Figure. 9 The fluorescence per OD.




Figure. 10 The fluorescence per particle.

2.

Flow Cytometry

We introduce a magic protein, anti-GFP nanobody, which is very small (only 13-kDa, 1.5nm 2.5nm) and high-affinity (0.59nM) camelid antibody to GFP[8]. So we can use its characteristic to improve our designs. We can fuse GFP to the C-terminus of interaction modules and to the N-terminus of HOTags, and fuse function modules to the C-terminus of anti-GFP nanobodies. Then, with the help of interaction between anti-GFP nanobodies and GFP, synthetic organelles will “welcome” function modules, expected functions can be realized. You may ask: How does anti-GFP nanobody improve the design? Firstly, it will not make the protein extremely large and will reduce the effect on the structure of function modules, which can ensure the quality of functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can enlarge the enrichment range of synthetic organelles. Thirdly, it is easy to regulate the expression of target proteins. So you can see, nanobodies may do better and give you a surprise!

Figure. 7 Interaction of anti-GFP nanobody and GFP
Conclusion

We artificially designed phase separation in cells and synthesized membraneless organelles. And the main work to synthesize an organelle is to fulfill phase separation in a cell, so we stress the importance of interactions and multivalency. For these two aspects, we gave our ideas and the feasibility was analyzed. At last, we proposed two ideas to implement functions. We believe that in the near future, “millions of dollars” will no longer be a dream!

References

[1] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
[2]https://static.igem.org/mediawiki/2018/e/ec/2018_InterLab_Flow_Cytometry_Protocol.pdf

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