Wet lab

Overview

After we finished the software, we decided to carry out an alpha test which involves the main functions of our software. Therefore we designed a circuit and build it up in a vector with the help of our software. Expression data are measured and can be used to validate and calibrate the software. Here we show the whole detailed process of the experiment.

Design our circuit

Search devices for our design

Edition of our circuit

The final design and our plasmids

Manage our experiments

Plan our experiment

We use our software to plan our experiment and schedule our time very well.

Result

Confirmation

We built applicable constructs using standard biobrick assembly and we introduced the three constructs into Escherichia coli strain DH5α cells. Photo.1, photo.2 show the confirmation result of the constructs by conducting PCR.

photo 1 20150827 ciy1-4 RBS+UVR8-tetR-Ter1-2 RBS+tetG 02 ciy

photo 2 qie p-r-t rbs

Fluorescence and OD600 data

Simulation

Experimental data

After the sequencing confirmation, we collected the data every hour for ten hours and the fluorescence and OD600 of the liquid cultures were measured using a plate reader. At the beginning, we used tetracycline which inhibits TetR repression. Chart.1, Chart.2 and Figure.1 Show the fluorescence and OD600 data

Discussion

Why we did not use the sunlight sensor as our input source anymore?

Before further experiment, we needed to ensure all the parts that we would use were in order so that we conducted a PCR for all the parts that we used after the propagation of the plasmid by transforming them which are in 2015 distribution kit into E.coli. Besides, we sent samples of all plasmids for the sequencing. Afterwards, we performed a sequence alignment between the sequencing result and the sequence which is provided by the registry. However, we only found one third of the sequence matched the official sequence in the UVR8-TetR sequencing result. Therefore, we decided to change an input source.

How did our circuit work without the sunlight sensor?

We decided to use the toggle switch by using tetracycline as an input. In the toggle switch, tetR represses ptet, and tet inhibits this repression so that ptet works. With cI inhibiting pcI and tetR being unable to inhibit ptet, the toggle switch is switched to YFP state. As our data above shows, yellow fluorescence goes up.

Doesn't tetracycline kill your bacteria?

To prevent this, the ptet-RBS-cI-RBS-YFP-terminator circuit is constructed in tetracycline resistance plasmid backbone.

How to explain the difference between simulation and experiment data?

We can see fluctuation in the data of the 2nd, 3rd and 4th hour. This may be caused by random error of the plate reader. The data curve went more steadily from 5th to 8th hour than the simulate one. That's because we only used one tube of culture for measurement. Every time we took out 200μ culture for measurement, some of the bacteria were lost, and the growth were also paused for a moment due to the low temperature. This influence is especially obvious in the first few hours, but the two curves would finally be closer to each other. From 8th to 10th hour, the data curve rose much more rapidly because in this period, the lost of bacteria caused by measurement can be neglected. As there were less bacteria in the earlier period, the resources and space made it possible for bacteria to grow more rapidly.

Testing

We did some tests, including installability test and running test, on Windows and MacOS X, to supplement the installability test on Linux that have been kindly done by judges.