Team:Marburg/Measurement
Aim
After working on the InterLab Study we asked ourselves, how much characterization is needed to fully describe a part or construct. With the InterLab Study, iGEM invited all the competing teams around the world to measure fluorescence from the same three genetic devices for GFP expression. The aim of the InterLab Study is to investigate the reproducibility of BioBrick characterization by collecting data of the same experiment from iGEM Teams around the world. Since standardization is an important aspect for synthetic biology, we wanted to show a way to make an overall and deep characterization of devices. For that we extended our InterLab Study project to a Measurement Study and characterized the promoters with diverse methods, in different constructs and chassis. Our goal is to create the best-characterized parts in the registry of biological parts. To achieve this goal, we used the plate reader, flow cytometry, proteomics and microscopy experiments to measure the fluorescence in single cells and in population. We introduced the devices into different strains, DH5alpha and the wild type MG1655. We also expressed RFP instead of GFP and normalized our data not only over the OD but also over the expressed RFP. We additionally showed the impact of evolution that can be seen in protein expression over time and quantified the noise of the gene expression. With this we contributed to a roadmap for characterization to the BioBrick registry and brought part characterization to the next level.
Project Design
We extended the Interlab Study constructs in order to be able to better characterize the promoter, and the expression on both single and population level. In order to see if the coding sequence of a gene has an influence on the expression level, we build a construct, where the GFP is replaced by an RFP. Additionally we wanted to normalize not only over the OD, but also over an internal standard. Therefore we placed a second gene with a strong promoter behind our GFP InterLab constructs and normalized over the other fluorescence. This double promoter-fluorescence gene construct was also used to measure the noise of our expression system. As not only the constructs themselves can lead to different expression levels but also the chassis, we used two common lab strains, Dh5alpha and MG1655 in order test our constructs. The expression levels were measured with a variety of different techniques and will allow a more comprehensive characterization and description of our constructs.
Results
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Outlook
In our follow up studies, we want to establish a standardization pipeline from construction till the provision of a data sheet. The registry of biological parts is the biggest collection of its kind, but many researchers that we have spoken to, hesitate using it. The reason for that is the lack of characterization and therefore a lack of quality. All iGEM Teams have to face this challenge and see it as a contribution to the field and the community of synthetic biology to provide a good characterization of their parts and construct. However, there is a strong need for standardization and characterization facilities in synthetic biology. As teams submit the BioBricks around the world, there should be a synthetic biology standardization facility in each continent, so that the standardization effort becomes an international call in synthetic biology. We as the iGEM Team Marburg will continue our work on part characterization. We want to extend our efforts further, for example on the RNA level, to have indications on all levels of protein biosynthesis about the expression of the fluorescent proteins. Much more work is needed to aim our goal of perfect characterization of standard biological parts.
Background
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Lessons Learned from Measurement Study
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