As the name of our project suggests, we hope that our team's contribution will help light iGEMer's stories in the future. We have made a lot of efforts in refining and improving existing parts. We also provide new design ideas and broaden the application of iGEM projects while working on the promotion of synthetic biology ideas.
In this year's iGEM project, we change our chassis into Bacillus subtilis, and with it came the challenge of transforming the strain. We tried two different electrotransformation protocols as well as three different transforming protocols using special medium and finally found the protocol provided by team LUM-Munich 2012 works best in our lab. This provides a stable and reproducible Bacillus subtilis transformation protocol for future iGEM teams that may use B. subtilis as their chassis.
This year we use plasmid pHY300PLK as a vector. To select and sequence the exogenous fragments introduced to this plasmid, we designed a pair of primers located upstream and downstream of the MCS on the plasmid backbone. See BBa_K4202048 and BBa_K4202047 to gain more information.
Firefly luciferases are currently being applied as analytical reagents and reporter genes for bioimaging and biosensors. Luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin, improving the luminescent signal of firefly luciferase. BBa_K325210 contains the coding sequence for a mutant light-emitting enzyme (luciferase) and luciferin regenerating enzyme (LRE) from the Japanese firefly. To provide more laboratory data for these enzymatic reactions, we measured the activity of these two enzymes in vitro experiments. We also completed kinetics measurements and KM determination for the luciferase.
We added documents of Part BBa_K325210 in its Registry Page based on our results. For the results and other details, see our Measurement page.
BBa_K4043004 is our improved promoter that can respond to sucrose concentration in the medium. We change its constitutive promoter and multiply the regulatory sequence, which leads to improving the degree of induction and making the induction more stable in most growing conditions theoretically.
BBa_K4202004 is a carbonic anhydrase coding sequence codon optimized for B. subtilis. Although the improved part didn't improve the carbonic anhydrase production, we confirmed the role of carbonic anhydrase and provided practical experience in the application of carbonic anhydrase for future iGEM teams.
For the results and other details, see our Improvement page.
We often use reporter genes (eg. luciferase and fluorescent proteins) in part characterization. However, existing fluorescence and luminescence measure devices mostly use photomultiplier tube as detector, which can only measure the light intensity in a small localized area and can’t the measure multiple samples with multiple channels at the same time. Moreover, the existing devices are also high cost. To address these problems and make fluorescence detection equipment available to every lab, we designed, built, and tested LviSense, a high-sensitivity camera based bioluminescence detector. Our new hardware is low-cost, and can sensitively detect concentration of fluorescent protein as well as the activity of luciferase. At the same time, LviSense has excellent heat preservation property, which maintains the bioluminescence reaction at a specific temperature or under the temperature cycle.
For more details, see our Hardware page.
Fig 1 LviSense
Fig 2 Hardware Design
To better implement the idea of iGEM into reality, this year we built a big synthetic biology platform of ZJU-China! It has integrated most of the resources of experimentation, education and entrepreneurship from the previous team, and also contributed to the subsequent iGEM Team.
See our integrated human practices page for more details.
Fig 3 Our communication platform
