Difference between revisions of "Team:DTU-Denmark/Design Measurement"
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| − | <img src="https://static.igem.org/mediawiki/2019/ | + | <figure> |
| + | <img style="padding:28px;" src="https://static.igem.org/mediawiki/2019/c/c1/T--DTU-Denmark--NewDesignDataRFP.svg" alt="The figure shows three curves visualising the exitation levels for mRFP1, mCherry EX, and moxGFP EM. mRFP1 and mCherry EX mostly overlap but while mCherry EX only peaks around 580 nm, mRFP additionally peaks around 500nm which overlaps with the moxGFP EM peak." class="safetyfirstimg"/> | ||
| + | <figcaption>Standard curves to interpolate our fluorescence data into a known concentration of a fluorescence standard is easily obtained using TexasRed (Sulforhodamine 101) [3]</figcaption> | ||
| + | </figure> | ||
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| − | <h2> | + | <h2>Choice of fluorescent protein</h2> |
| − | <p> | + | <p>For testing the synthetic promoters, the red fluorescent protein <a href=”http://parts.igem.org/Part:BBa_J06504” target=”_blank”>mCherry</a> was chosen as the fluorescent reporter. mCherry was used for a number of reasons, including: |
| + | <ol> | ||
| + | <li>mCherry has been proven to work both in <i>E.coli</i> and filamentous fungi. This enables our test device to be used for screening for correct insertion of synthetic promoters in <i>E.coli</i>, as the expression of mCherry can be seen with the naked eye.</li> | ||
| + | <li>Compared to mRFP such as [part number for J04450], the maturation time is short, which is beneficial when analysing the promoter dynamics.[2] </li> | ||
| + | <li>Compared to mRFP, mCherry does not have a peak in absorbance around 500 nm. For mRFP, this peak means that it would absorb part of the signal from GFP, thus obscuring the result if we are to introduce a double fluorescence calibration system as described in <a href=”https://2019.igem.org/Team:DTU-Denmark/Design_Plasmid#FutureDesign” target=”_blank”>Future design</a> and the figure below. </li> | ||
| + | <li>Fluorescence standards such as TexasRed are easy to obtain, and have a nice overlap in spectra with mCherry. Furthermore, the iGEM measurement committee has also <a href=”https://2019.igem.org/Measurement/Resources#fluor_proteins”>recommended the use of TexasRed</a> as a chemical standard for mCherry.</li> | ||
| + | </ol> | ||
</p> | </p> | ||
Revision as of 16:27, 21 October 2019
Measurement Design
Measuring promoters
A lot of considerations have gone into how to reliably measure the performance of our promoters. We decided that the primary method of measuring expression from our promoters would be fluorescence reading as we wanted to test the promoters in three different scales: Microplate scale (1-1.5 mL cultures), shake flask scale (200 mL cultures), and bioreactor scale (1 L cultures), also described on the Demonstrate page.
Using a fluorescent reporter
By using a fluorescent reporter gene to measure promoter output, we were able to use a micro bioreactor (BioLector®, M2Plabs) for our microplate scale experiments. The BioLector is able to continuously measure cell density and fluorescence while cell cultures are growing, thus giving us data with a good enough temporal resolution to analyze the dynamics of the different LEAP promoters. Additionally, expressing a fluorescent protein that produces a relatively stable signal is a big advantage compared to having the signal heavily influenced by external factors such as temperature when purifying enzymes for subsequent assays. This is especially important when working with larger experiments, since the frequent measurements we did would have been impractical to carry out with a less stable protein if we were to get the time resolution required for detailed analysis.
We did consider using expression of enzymes such as β-glucuronidase to test our synthetic promoters, since these are known to be secreted by A. niger. β-glucuronidase has also been used in previous studies on promoter activity in A. niger[1], but despite these advantages for the enzyme, we decided the benefits from using fluorescent proteins to get continuous measurements from the BioLector would be greater than the slight disadvantages posed by not having the fluorescent proteins exported from the cells.
Choice of fluorescent protein
For testing the synthetic promoters, the red fluorescent protein mCherry was chosen as the fluorescent reporter. mCherry was used for a number of reasons, including:
- mCherry has been proven to work both in E.coli and filamentous fungi. This enables our test device to be used for screening for correct insertion of synthetic promoters in E.coli, as the expression of mCherry can be seen with the naked eye.
- Compared to mRFP such as [part number for J04450], the maturation time is short, which is beneficial when analysing the promoter dynamics.[2]
- Compared to mRFP, mCherry does not have a peak in absorbance around 500 nm. For mRFP, this peak means that it would absorb part of the signal from GFP, thus obscuring the result if we are to introduce a double fluorescence calibration system as described in Future design and the figure below.
- Fluorescence standards such as TexasRed are easy to obtain, and have a nice overlap in spectra with mCherry. Furthermore, the iGEM measurement committee has also recommended the use of TexasRed as a chemical standard for mCherry.
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