Biosafety Motivation

Abstract

Beside the research into the project it is important to collaborate with other iGEM teams to support them. This support can be carried out in different ways, for example an exchange via a skype interview or characterize the other part of the other team. In the following description we list our collaborations between us and other teams.

iGEM-Team York_UK

Because of the fact that both teams do research into microbial fuel cells we get in touch and talked about our projects. We get to know that York had problems with cloning the mtrCAB cluster and invested a lot of time to solve this problems. Because of the fact that time was running we decided to help York. We already designed our MFC and tested different constructions which one is the best to use. We constructed a MFC specially adapted to the requirements of York and wrote an instruction how to operate with the MFC. In addition we put nafion membranes, gaskets, carbon electrodes and injectors into a package and send it to York. We hope that this support helped York to generate good results and finish their project.

iGEM-Team NRP-UEA-Norwich

The iGEM-Team from Norwich developed a biosensor which enable the identification of antimycin-producing strains of streptomyctes. Therefore they asked us to send them soil samples from Bielefeld. Of course we took samples of soil and send them to Norwich. The interesting soil samples of Bielefeld are shown below. We hope that our soil samples helped Norwich.

Figure 1: Sample No. 33

Figure 2: Sample No. 34

Figure 3: Soil from which the samples were taken

iGEM-Team UC Davis

The iGEM-Team UC-Davis 2013 wants to develop and implement an experimental data depot for biobrick characterization data and kindly asked us to collaborate with them. We tested their promoter characterization protocols on the Anderson promoter collection J230100-J230119. The plasmid backbone of these biobricks J61002 is very suitable for promoter insertion between the XbaI and SpeI sites, which places the promoter upstream of a red fluorescent protein (RFP)gene. Through the promoter, RFP is expressed more frequently or less, while the promoter strength can easily be characterized via fluorescence measurements.


Our team focused mainly on the second half of the collection, which comprises promoters with the below listed strengths.

We carried out an extended, 18-hour experiment, monitoring cell density through absorbance measurement and promoter strength by way of fluorescence emission. The results are shown subsequently.

Figure1: Absorbance at 700 nm of E. coli DH5α-strains with transformed Anderson promoter familiy members

Please note that we monitored the optical density at 700 nm wavelength, because the UC-Davis team was concerned about interferences of OD600 measurements with the fluorescence measurements.
To determine the relative strength of the used promoters we looked at the fluorescence signal of RFP. With increasing promoter strength, transcription should be enhanced and more RFP will be expressed. This results in a higher fluorescence signal.

Figure1: Fluorescence emission at 615 nm of the stronger Anderson promoter family members (excitation at 588 nm). The values in brackets indicate the relative promoter strength compared to J61100, which was given the value 1.

Figure1: Fluorescence emission at 615 nm of the medium Anderson promoter family members (excitation at 588 nm). The values in brackets indicate the relative promoter strength compared to J61100, which was given the value 1.

Figure1: Fluorescence emission at 615 nm of the weaker Anderson promoter family members (excitation at 588 nm). The values in brackets indicate the relative promoter strength compared to J61100, which was given the value 1.

The results show very clearly, that strains containing a plasmid with a stronger promoter are fluorescing earlier than those with medium and weak promoters. Also, the fluorescence intensity is significantly lower for weak promoters in comparison to the stronger ones. The DH5α-strain with an inherent J23101 plasmid (relative strength 0.7) did surprisingly not fluoresce the strongest, which is why a problem with plasmid stability in this strain could be assumed. Also, the unmeasured J23119 biobrick seems to hold a promoter of medium strength.

The widely plausible results show, that the protocols for promoter biobrick characterization work quite well. Comparisons to experimental results from all over the world can be carried out using this method and promise a well characterized and frequently validated parts registry.

This collaboration was a great chance to communicate and discuss data in an international outreach and taught us a lot about other teams work routines and standards. It was a pleasure to contribute in their project to find a way to effectively share complex experimental data and to work out routines for biobrick characterization