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: the high-copy plasmid [http://parts.igem.org/Part:pSB1C3 pSB1C3] (100-300 copies/cell) and the low-to-medium-copy plasmid [http://parts.igem.org/Part:pSB3K3 pSB3K3] (20-30 copies/cell). 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 verification PCR-positive clones were sequenced: device two and device three

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 intact cells during the measurement. After the incubation, 1 µl of each sample was diluted in 200 µl phosphate buffered saline solution and then evaluated via flow cytometry. In order to generate meaningful results the described measurements were perfomed three times with technical replicates.

For the analysis, all events were gated for a signal in the red channel (which corresponds to intact 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

The results generated here show a 10-fold higher expression read out with GFP for [http://parts.igem.org/Part:BBa_K823005 BBa_K823005] compared to [http://parts.igem.org/Part:BBa_K823012 BBa_K823012]. Having a closer look at the [http://parts.igem.org/Part:BBa_I20260 BBa_I20260] device, the utilization of the low to medium-copy plasmid [http://parts.igem.org/Part:pSB3K3 pSB3K3] explains the lower expression of GFP compared to [http://parts.igem.org/Part:BBa_K823005 BBa_K823005], despite containing the same insert.

Outlook

The LMU-iGEM team is looking forward to the presentation of iGEM HQ results, where we hope to see a mixture of different methods utilized - hopefully with similar outcomes. We are curious how the absolute and relative results might differ; this might be a milestone towards ensuring reproducibility of scientific data produced by a huge number of different labs and methods.

Collaboration with Team Groningen

On August 1st, not only the collaboration with the Rathenau Instituut but also the cooperation with iGEM Team Groningen, was kicked off. During the first group chat with the Rathenau Institute, we were introduced to members of the iGEM Team Groningen that had with their 'Lacto Aid' a quite similar project idea to our BaKillus. As we pursue the same idea but work with different chassis, different targets and aim for different applications, we decided not to see them as rivals but rather use that chance to learn from their experiences and also support them, when help is required. From then on, we held regular Skype meetings and supported each other by exchanging views and thoughts about the feasibility and social desirability of our applications inspired by the guidelines of the Rathenau scenarios. Encouraged by this first steps, we also helped each other out in the lab.

Efforts of team Groningen

As our BaKillus targets Staphylococcus aureus, but we do not have access to S2 laboratories, and thus unfortunately cannot experiment with this bacterium. Our collaboration partner kindly provided us help by testing the subtilin producer Bacillus subtilis ATCC6633 in a spot-on-lawn assay on different S. aureus strains, including MRSA. Fortunately, the results could prove subtilin has inhibitory impact on S.aureus and thus, serves as excellent killing strategy for our project.

Fig. 1. Spot-on-lawn assay with the subtilin producer on three different S. aureus lawns

The results are part of our killing subproject and can be seen here or detailed information downloaded here.

Work done in our Lab

On Monday 13th of October we received parts to be evaluated in Bacillus subtilis from the iGEM Team Groningen. The goal was to evaluate Promoters fused to a super-folded Green Fluorescent Protein (sfGFP) towards their activity in B. subtilis. In order to do so we had to bring the BioBricks into our integrative BioBrick compatible backbone [http://parts.igem.org/Part:BBa_J179000 pSB1C]. The final analysis was done using fluorescent-activated cell sorting (FACS). The results of the strains carrying the Promoter-sfGFP insertion were compared to the B. subtilis W168 strain.

The following Promoters were analyzed: [http://parts.igem.org/Part:BBa_K1033219 CP1], [http://parts.igem.org/Part:BBa_K1033221 CP11], [http://parts.igem.org/Part:BBa_K1033222 CP29], [http://parts.igem.org/Part:BBa_K1033223 CP30], [http://parts.igem.org/Part:BBa_K1033224 CP41], [http://parts.igem.org/Part:BBa_K1033225 CP44]

The data achieved via FACS shows no change in distribution of FL1-A intensity. This strongly inidcates, that the promoter-sfGFP constructs are not active when integrated into the B. subtilis chromosome. This might however be not only due to an lack of Promoter activity. To further invesitgate a quantitative assay using promoter-lux fusions should be carried out.

Table 1: FACS Data

A proposal of the German iGEM Teams concerning Intellectual Property

During the meetup of the German iGEM teams from 23rd to 25th May a workshop also 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 the parts registry remains unclear and that there are parts (e.g. [http://parts.igem.org/Part:BBa_K180009 BBa_K180009]) where it only becomes clear upon a closer that the rights are company–owned. This issue (that the legal status of parts in the registry remains uncertain) is also mentioned in a recent article published by Nature [1]:

"[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 the 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 relevant. It would also raise awareness to the topic of the legal status of parts. This could lead to a debate which could further promote the idea of open source, ideally. At the same time we hope that the 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

References:

• [http://www.nature.com/news/synthetic-biology-cultural-divide-1.15149| Bryn Nelson ‘Synthetic Biology: Cultural Divide ’, Nature 509, 152–154, 08 May 2014]

Collaboration with Team Virginia

   
    
        
    
     
    
        

            
        
    
The global synthetic biology awareness and acceptance survey designed by the Virginia iGEM-team was a great opportunity to generate a lot of data, 
since more than 40 teams participated!