Difference between revisions of "Team:Munich/Measurement"

Genetic modification of microorganisms is an essential method in the field of synthetic biology. By ‘thinking outside of the cell’ a cell free transcription/translation (TX-TL) system has emerged to be a true alternative by expanding the capabilities of a natural biological system from in vivo to in vitro ¹. While commercialized TX-TL systems exhibit good reproducible results, they are only available at high cost. Contrary to commercial systems, home-made TX-TL systems are affordable but come with the drawback of high batch-to-batch variations after time-consuming preparation steps. Further investigations in the optimization of the protocol for TX-TL preparation are necessary to create an easily accessible platform for the research community. Our iGEM team developed a protocol for the easy and high-quality production of a cell extract, which can be reproduced in any laboratory without the need for expensive equipment. This TX-TL system can be used to measure protein expression, genetic circuit performance and other synthetic biology tools.

To obtain a high quality cell extract, we focused on the optimization of several key factors: The culture conditions were optimized by screening for the optimal cell density to harvest cells. Assessing several different lysis methods, we identified sonication as the cheapest, most accessible and reproducible way for the production of our TX-TL protein expression system. Furthermore, the upscaling of TX-TL preparation was demonstrated by using a bioreactor for cell cultivation. Elaborate steps like the removal of the endotoxin Lipid A from the TX-TL were rendered obsolete by designing a suitable mutant strain for TX-TL preparation. For this purpose, TX-TL was prepared with a strain lacking the msbB gene to disrupt the lipid A biosynthesis pathway. By this the lipid A concentration could be lowered by a factor of 49 compared to the cell extract prepared using the wild-type strain (0.06 EU/mL).

The produced TX-TL went through further quality control checkpoints consisting of measuring protein content, as well as an activity assay via expression level analysis. The protein concentration was determined by using a commercial BCA protein assay kit. Compared to the commercial TX-TL, our home-made TX-TL shows a similar protein content of about 16 mg/mL.

However, a distinct and functional quality control requires the determination of the protein expression rate. This was done by expressing fluorescent proteins (GFP, YFP, RFP, CFP) and recording the corresponding signal using standard lab equipment like a plate reader. Extending the mRNA of the fluorescent protein mTurquoise2 (mTQ2) with the sequence for the malachite green aptamer (MG), we planned to determine the transcription and translation rate in parallel. Unfortunately, we could not express a functional fused mTQ2-MG and instead measured the transcription and translation separately by using the single components on two different plasmids.

Moreover, mTQ2 showed the fastest maturation time and lowest signal-to-noise ratio of the tested fluorescent proteins. Due to its long fluorescent lifetimes (> 3.7 ns) and high quantum yields (> 0.8) it delivers significant results for the quality control of the translation performance of our TX-TL⁴.

The expression of mTurquoise2 showed a five times higher fluorescent signal in our home-made TX-TL than in the commercialized TX-TL and therefore confirms that our optimized protocol produces cell extract of high translation efficiency. Although the overall protein expression rate can be observed with this method, the transcription rate has to be determined using a different approach.

For an excellent quality control, the transcription efficiency of our home made TX-TL was analyzed separately. We used a plasmid containing a malachite green aptamer downstream of a T7 promoter. The malachite green aptamer, a small RNA transcript, is able to bind a specific ligand, malachite green and enhances its initial fluorescence >2000 fold⁵.

By observing the fluorescence signal over time the transcription levels of different TX-TL batches can be compared.

These results prove our home-made TX-TL as a high-quality expression platform regarding transcription as well as translation. Thus, it forms a perfect platform for testing various biological parts listed in the iGEM Registry. The standardized expression system allows a more reliable comparison of BioBricks for iGEM teams around the world.

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