Team:Chalmers-Gothenburg/Contribution

Summary

We have made three worthwhile contributions worth to mention; we have helped characterize three different ribosome binding sites (RBS), one weak, one medium and one strong. Secondly, we have laid out a foundation for hosting the Nordic iGEM conference for future teams. Lastly, we also contributed with a code to make it easier and faster to upload files to the iGEM database and convert it into usable HTML code. We believe that all these contributions will facilitate future iGEM teams.

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RBS

NiC Slack

Code

Page Content Background Aims Experimental Design Methodology Results Discussion References

Characterization of ribosome binding sites

Background

A ribosome binding site is a short DNA sequence (10-20 base pairs) that when transcribed into RNA along with a coding sequence is responsible for binding to the ribosome and initiating translation of the mRNA, influencing the amount of protein transcribed per mRNA fragment. By swapping out RBS sites, expression levels in bacteria can be altered, and good knowledge of different sites is important.

Aims

We set out to characterize three previously reported RBS sites, BBa_K3252027 which was reported to be a very weak RBS, BBa_K3252026 which was reported to be a medium strength RBS and BBa_J15001, which has been reported as being a very strong RBS.

Experimental design

Due to the short length of RBS, we decided to utilise a modified PCR primer method, whereby the sfGFP-containing plasmid pYTK001 from the MoClo kit was amplified by a forward and reverse primer pair containing an overhang with the RBS, designed as that it blocked and overwrote the natural RBS present, replacing the native RBS.

Methodology

pYTK001 was mixed with a custom forward/reverse primer pair and PCR reaction carried out according to protocol using Phusion polymerase. The produced solution was purified using gel purification followed by gel purification kit from Thermo Fisher, and the resultant linear fragment was cyclised using Gibson assembly protocols, followed by transformation into competent E.coli strain DH5-alpha. The plated cells were inoculated, grown overnight and plasmids extracted using Thermo Fisher plasmid miniprep kit and protocol. From the extracted plasmids a sample was sent to sequencing, while the remainder was used to transform again, and following overnight growth, inoculated again, followed by equalizing the OD of each sample. Alongside a positive control consisting of unmodified pYTK001 sfGFP-containing plasmid, a negative control of transformed pYTK002 (without GFP) in DH5-alpha E.coli, the fluorescence was measured with three replicates of each RBS using a plate reader and the strength of each RBS was measured.

Results

The RBS were measured using plate reader and the data was then collected and processed to reveal the data provided in figure 1.

Figure 1: Fluorescence expression dependent on RBS strength.

RBS weak showed slightly more activity than the negative control, while the medium RBS was somewhat stronger than the weak RBS. The strong RBS showed the same values as the negative control and the positive control had many orders of magnitude more fluorescence than any of the other RBS tested. Sequencing confirmed the RBS to be correct and without mutation, and no other errors were noted.

Discussion

The results were quite contradictory to what we were expecting, particularly with the strong RBS showing expression levels equal to that of the negative control, which suggests that the strong RBS completely disables either transcription or translation of the sfGFP gene. Alongside this, the weak RBS result was expected, and the medium RBS was on the lower side, but still showing a stronger expression level than the weak RBS and much higher than the negative control. It is also not believed that the very high response of the positive control is an error or cause for concern, since the pYTK001 plasmid was not modified and is a well-characterized part of the MoClo kit (1). All samples with their three replicates were sequenced and no mutations or other errors were noted in the RBS and sfGFP sequence, so it is unlikely that errors in assembly or random mutations are responsible for the poor results from RBS strong and RBS medium. We invite future iGEM teams to characterize these ribosome binding sites as to provide further insight on their function.

References

1: Lee ME, DeLoache WC, Cervantes B, Dueber JE. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth Biol. 2015 Sep 18;4(9):975-86. doi: 10.1021/sb500366v. Epub 2015 May 1. PMID: 25871405.

Page Content NiC

Nordic iGEM conference (NiC)

When organizing the Nordic iGEM Conference (NiC) we realised that there was a general lack of documentation that would help the transition between previous hosting teams and the new ones. Traditionally, a competition is held every year for 'Best Presentation' and the winning team would receive the honour of hosting NiC the coming year. Unfortunately, for various reasons, the winning team from NiC 2020 announced that they would be unable to host NiC 2021. As our team were already excited about organizing a similar online event, we realised we had to make sure this fine tradition of having meet-up between all the Nordic iGEM team was continued! However, we had difficulties finding information about previous conferences. What should the content be? Should we invite guest speakers? How was the winners supposed to be decided?

With this in regard we created a Slack channel specifically for NiC where the Nordic iGEM teams would be able to communicate, share ideas and documents, and find inspiration in each others projects. The idea was to make it easier to find information about NiC and specifically how to host NiC for future teams. We also made transitional documents available with tips & trix, general guidelines and inspirational materials that we hope future hosting teams will continue to update so that less time have to be spent on planning the event from scratch.

Page Content code

From filename to HTML code

One thing we felt was overly time consuming was uploading files and documents to the iGEM database, as well as converting the links to corresponding HTML code. As such, to make our life a tiny bit easier we created a code which first made the named the files to be uploaded, and took the made links to a predefined HTML code through a template. This sped up the process considerably, and as such we thought we could share it and hope future iGEM teams might feel it can come in handy.

The code is written in MATLAB which is a one of the new sponsors for iGEM, as such all iGEM teams can use the code.

MATLAB Code

Download zip File