BOX

Aim

This year, iGEM invited all the competing teams around the world to measure fluorescence from the same three genetic devices for GFP expression. Teams all around the world measured the fluorescence of the devices at on OD of 0.5. The reason for this study is to show the variability between labs and to create a so defined standard for constructs used in synthetic biology. Since standardization is one of the most important parts in synthetic biology, we wanted to show a way to make an overall characterization of devices. For that we extended the InterLab Study in our Measurement Study and characterized the promoters with diverse methods and in different constructs. We used plate reader, flow cytometry, proteomics and microscopy to measure the fluorescence in single cells and in populations. We introduced the devices in two different strains: DH5alpha and the wild type MG1655. We expressed RFP instead of GFP and normalized our data not only over the OD but also in our double constructs also to the expressed RFP in our double constructs. We also showed the impact of evolution that can be seen in protein expression over time and quantified the noise of gene expression.

Project Design

We extended the Interlab Study constructs (Fig. 1) in order to be able to better characterize the promoter, and the expression on both single cell and population level. In order to see whether 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 (Fig. 2). Additionally we wanted to normalize not only to the OD, but also to an internal standard. Therefore we placed in two constructs a second fluorescent gene (RFP) with a strong promoter upstream of our GFP InterLab constructs 1 and 2 (Fig. 3). 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

Platereader

The platereader results from the stationary phase of our constructs in MG1655 and DH5alpha (Fig. 4) are comparable to the ones we obtained for the Interlab Study. Like previously described, we diluted an over night culture to an OD of 0.5 and depending on the strain measured the fluorescence intensity of GFP, RFP or both. As described on the registry page of the constitutive promoter family we used construct 1 to 3 with decreasing promoter activity. In most cases, the normalized promoter activity was higher in MG1655 than in DH5alpha. Taken together, we show that the chassis plays an important role for the characterization of a construct. In order to provide a comprehensive characterization/analysis we used both, the commonly used cloning strain DH5alpha as well as the wildtype strain MG1655 for our further studies.

The timelapse measurement of our constructs reveals that fluorescence intensity increases over time. The used fluorescent proteins are not fused to a degradation tag, which may have led to an accumulation of GFP and RFP and hence to an increase of fluorescence intensity. In accordance with the one-point measurements in the plate reader, we observed a variation between the two different chassis. Also the expression levels and the ratios of fluorescence intensities are consistent.

Flow Cytometry

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Microscopy

By applying single cell microscopy we could analyze the phenotypical as well as the gene expression variability of cells for constructs 1-3. Cells carrying our construct 1- with a strong promoter- are elongated and in some cases stop dividing, indicating that cells are stressed likely by the burden of the construct. Cells carrying the weaker promoter of construct 2 and 3 did not reveal any changes in cell size and shape. Additionally, in order to quantitatively analyze the fluorescence intensity of the cells, we applied the computer algorithm “microbeTracker” (Garner, 2011) with which cells were analyzed and the results are summarized in figure XXXX. Briefly, cells carrying construct 1 were high in fluorescence intensity and the exposure time needs more precise adjustment; and cells carrying construct 2 showed a high variability in fluorescence intensity. The best suitable promoter for further analysis for a homogeneous expression profile is construct 3. For this construct we fused the weakest promoter to GFP and observed a Gaussian distribution in fluorescence intensity. Taken together, we analyzed our InterLab constructs by microscopy and have now more information about the expression variability. This can be an important characteristic for possible applications.

Noise

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Evolution

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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

In synthetic biology, it is important that part characterization is consistent between different labs to be able to create well-defined standard parts whose behavior is predictable. One of the fundamental principles in Synthetic Biology is engineering. But different from electrical or mechanical engineering, Biology engineering makes use of life itself. Our biological constructs are self-replicating and there is an interaction between our circuits and the chassis that we choose to express them in. Also most biological Parts are not as well defined and characterized as in other engineering disciplines. Because of that, one of the most challenging parts in the transition from science to an engineering field is to define standards. Every engineering discipline is very fond of standardization. In the classical mechanical engineering, standards are used to provide sufficient information about a part and the defined standards lead to an abstraction of an element’s behavior and the simplification of the design. Hence parts can be treated as a black box, which can easily be combined with others. The most common example for standardization in synthetic biology is the BioBrick standard generated by the iGEM competition. Another example is the recently introduced Standard European Vector Architecture. But as we can already see with this example, there is no uniform concept of DNA part standards. Besides the introduction of a common standard also the characterization of parts should be standardized. Both the BioBricks and the Standard European Vector Architecture are only construction standards. Developing a characterization standard is even harder to reach, as Biology is a complex science and each part and construct behaves differently in the different chassis. It is hence hard to define characterization procedures and protocols and to be able to compare them. One way of approaching the characterization is to test the different behaviors of a construct. In our measurement project we tried to follow a holistic approach in order characterize a part as complete as possible and to introduce a new standards of how to characterize parts.

Lessons Learned from Measurement Study

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