• Team
  • Team
  • Financing
  • Collaborations
  • Attributions
• Project
  • Problem
  • BaKillus
    • sensing
    • adhesion
    • killing
    • suicide
  • Bacillus BioBrickBox 2.0
  • Modeling
  • B. subtilis
• Results
  • Results
  • Registry
  • Judging
• Notebook
  • Safety
  • Protocols
• Application
  • Diagnosis & Treatment
  • Pretest
  • Legal Issues
  • Financing & Intellectual Property
  • Expertise
• Human Practice
  • Meet ups
  • iGEM@school
  • SynBio Day
  • Panel Discussion
  • Rathenau Instituut

Collaborations

Collaborations and the exchange of knowledge were important aspects during our iGEM participation. Therefore we had a intensive collaboration with the iGEM team Groningen (Netherlands) and laid a high priority on the exchange with the Rathenau Institute also located in the Netherlands. In addition we also took part in the Interlab Study of this year´s iGEM competition.

Interlab Study

Background:

The aim of the Interlab study was to evaluate three devices consisting of different Anderson promoters fused to GFP in two different backbones: pSB1C3 and pSB3K3. The devices were distributed by iGEM and the flourescence evaluation method was free of choice. For more details check: Interlab Study@iGEM

The devices:

• Device one: BBa_I20260 consisting of J23101-B0032-E0040-B0015 in the backbone pSB3K3
  

Status: ready for evaluation

• Device two: BBa_J23101 + BBa_E0240, each in the backbone pSB1C3

Status: cloning before evaluation

• Device three: BBa_J23115 + BBa_E0240, each in the backbone pSB1C3

Status: cloning before evaluation

The cloning:

Devices two and three were built from their individual parts. The plasmids containing the promotors (BBa_J23101 and BBa_J23115) were cut open with SpeI-HF and PstI-HF restriction enzymes. The XbaI and PstI-HF digested GFP (BBa_E0240) was then ligated downstream of the promotors. Both devices were transformed into DH5α, plated out on chloramphenicol [35mg/µl] LB-agar plates and tested via colony-PCR to identify positive clones. For clarification PCR-positive clones were sequenced: BBa_J23101 + BBa_E0240.txt BBa_J23115 + BBa_E0240.txt

Flow cytometry analysis:

For each measurement 50 µl of an overnight culture were first incubated with 1 µl of the membrane stain FM4-64 (final concentration: 4ng/µl) at 37°C for 60 minutes. This is necessary to differentiate between dust or cell particles and cells during the measurement. After the incubation, 1 µl of each sample was diluted in 200 µl phosphate buffered saline solution and then evaluated by flow cytometry. In order to generate meaningful results the described measurement was perfomed three times with technical replicates.

For the analysis, all events were gated for a signal in the red channel (these are the actual cells). For those events, the gfp-fluorescence was measured in fluorescence units. The graph below shows the avarage of the geometric mean of the gfp signals from 3 experiments. The empty DH5α strain was used as control for auto fluorescence of the cells.

For details please check our Interlab study submission: LMU-Munich Interlab form

Results:

The results indicate that the promoter BBa_K823005 shows the highest expression of GFP, followed by the device BBa_I20260 (BBa_K823005 + B0032). The lowest GFP expression was measured with the device carrying BBa_K823012.

Discussion

Regarding the measurements from the LMU-iGEM Team of 2012 ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K823012 here]) our generated results go hand in hand with those from 2012 where the activity of several Anderson promoters were measured in B. subtilis. The Anderson promoter BBa_K823005 showed back then a 5 fold higher (at its maxima) LUMI-read out compared to the BBa_K823012.
The in 2014 generated results in E. coli show even a 10 fold higher expression read out with GFP fusion. Having a closer look at the BBa_I20260 device, the insertion of a weak RBS binding site (as described [http://parts.igem.org/Part:BBa_B0032 here]) might explain the lower expression of GFP in this device. In addition, the different backbone might also have an influence on the expression level and should be closer investigated for further conclusions.

Outlook

The LMU-iGEM team is looking forward to the presentation of iGEM HQ results, were we hope to see a mix of different used methods - hopefully with similar outcome. We are curious how the absolute and relative results might differ; this might be a milestone towards reproducibility of scientific data in a huge number of labs.

Collaboration with Team Groningen

A proposal of the German iGEM Teams concerning Intellectual Property

During the meetup of the German iGEM teams from 23rd to 25th May also a workshop took place in which amongst others we discussed the topic of bioethics. Moral questions were addressed, regarding the value of life and human influence on it, as well as questions dealing with the possible socioeconomic effects of synthetic biology.

Especially the topic of an open source vs. patent controlled field accounted for a large part of the discussion. During the discussion one student brought up the point that the legal status of parts in registry remains unclear and that there are parts (e.g. [http://parts.igem.org/Part:BBa_K180009 BBa_K180009]) where only upon a closer look it becomes clear that the rights are company–owned. The issue that the legal status of parts in the registry remains uncertain is also mentioned in a recent article published by Nature ([http://www.nature.com/news/synthetic-biology-cultural-divide-1.15149 Bryn Nelson ‘Synthetic Biology: Cultural Divide ’, Nature 509, 152–154, 08 May 2014]) :

"[N]o one can say with any certainty how many of these parts are themselves entirely free of patent claims."

We, the German iGEM teams, therefore like to suggest the addition of a new feature to the parts registry:

A dedicated data field of license information for each BioBrick part.

For the implementation, we propose to introduce two new fields to BioBrick part entries in the registry:

1. A string property "LicenseInfo"
2. A traffic light property (grey, green, yellow, red) to indicate the level of legal protection (unknown, BPA-like, free for research purposes, heavily protected)

Implementing this feature would in our opinion further clarify and extend the parts info, provide a machine-readable format and thus improve future entries. With the emerging Entrepreneurship track and applications getting closer to industrial realization, the legal status becomes more and more important. Also it would raise awareness to the topic of the legal status of parts, leading to a debate which could further promote the idea of open source. At the same time we hope that examination of most parts will show that they are indeed free of restrictive legal protections.

The German iGEM Teams,

Aachen Berlin Bielefeld-CeBiTec Braunschweig Freiburg Goettingen Hannover Heidelberg LMU-Munich Marburg Saarland Tuebingen TU Darmstadt

Collaboration with Team Virginia