Contribution

Part Measurement

To take the responsibility of developing synthetic biology and create a useful tool for future iGEM teams, this year we focused on the part BBa_K540001, Prcn [1]. We did some complementary kinetic measurements on Prcn and constructed a derived new promoter with similar function but higher efficiency. We added documents of Part BBa_K540001 in its Registry Page based on our results.

Prcn, in the part BBa_K540001, is a promoter that can be activated by cobalt. The RcnR repressor binds to the promoter in the absence of cobalt. When sufficient cobalt molecules accumulate in the cell cytoplasm, the cobalt ions bind to the RcnR repressor and prevent its attachment to the rcn promoter.

It was reported that rcnA and rcnR gene products have a connection with nickel, cobalt and iron homeostasis [2]. Thus we hypothesised that Prcn could potentially be regulated by nickel and even other metal ions. Meanwhile, the interaction between treating time, concentration of cobalt and efficacy of Prcn was still not well studied. To verify our hypothesis and construct a time-concentration-response curve, we did comprehensive measurement work based on constructed Prcn. And according to the experiment result, we also introduced a general framework of statistical-driven procedure for part kinetics characterization.

For the results and other details, see our Measurement page.

Troubleshooting

In our measurement and improvement experiment, we document unexpected results. We addresses the importance of aerobic environment when culturing bacteria, which might be helpful for future iGEM teams. For more details, see module "Lessons and hint from unexpected results" in our Measurement page and module "Results" in our Improve page.

Expmeasure – A Software for Part Characterization

Our measurement work did not stop at the characterization of a specific biological part. Statistical tests are powerful tools for kinetic research. Unfortunately, many of them are not correctly collected and used in a comprehensive way to assess the reliability of dataset generated by wet experiments, not along to provide guidelines for further experiments.

We developed a framework of statistical-driven procedure for part kinetics characterization. We constructed new summary statistics in order to provide implications for laboratory work and performed corresponding simulations for validation. We also integrated other methods in our framework as a potential way to improve accuracy and reproducibility of part characterization.

Based on this framework, we developed a software called Expmeasure. This R package provides a wide range of statistical analyses for part characterization, ranging from part kinetics measurement to differential expression analysis. Examples of how Expmeasure can help iGEM teams to identify potential trends, outliers, and 'junk data' in their dataset are given in the user manual based on our experimental work. A workflow of analysis, data input, address of potential error messages, and a quick-start example are well documented in the user manual. Expmeasure adopts a graphic-based interface, which allows any iGEM teams to perform comprehensive data analysis without coding.

For more details, see our Measurement and Software page.

Our Hardware

Transplantable impedance detection kit

We have packaged the detection system of our hardware work into a transplantable impedance detection kit. The kit mainly consists of three parts: the portable electrochemical detection station, the chip, and the controlling software.

The portable electrochemical detection station

This device was developed on the Arduino and the impedance expansion board we designed. The schematic blueprint of the expansion board is as follows: Fig. 1

Fig.1 Lay out of the board design

After being laid out (For example using Altium Designer), the circuit above can be printed and subsequently assembled (See Fig. 2).

Fig.2 a) The lay out of the circuit b) The expansion board after assembling

The shell

The shell of portable detection was made by laser cutting acrylic plate, the square opening at the front and rear was for the interface and the battery (See Fig 3). We have had our shell AutoCAD file and the circuit board layout file uploaded on Github, Please visit this page. You can use the AutoCAD file to reproduce the shell on any laser etching machine.

Figure 3. The layout of the shell in AutoCAD

For more details, see our Hardware page. You can also see some details about our APP in the Software page.

The Arduino controlling program and software codes

Please visit our project on this Github page

References

[1] Part:BBa K540001 - parts.igem.org

[2] Koch, D., Nies, D. H., Grass, G. (2007) The RcnRA (YohLM) system of Escherichia coli: A connection between nickel, cobalt and iron homeostasis. BioMetals, 20, 759–771. https://doi.org/10.1007/s10534-006-9039-6