Difference between revisions of "Team:DTU-Denmark/Design Measurement"
LouiseAlmer (Talk | contribs) |
|||
| Line 153: | Line 153: | ||
</div> | </div> | ||
| − | + | </div> | |
<div class="row flex-center sm-no-flex"> | <div class="row flex-center sm-no-flex"> | ||
Revision as of 16:20, 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.
Comming soon
Text will also be comming soon.
coming soon
more text coming soon
Comming soon
Text will also be comming soon.
Sources here will also come soon
