Difference between revisions of "Part:BBa K3046009"

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	<img style="width: 60%; padding:28px;" src="https://2019.igem.org/wiki/images/9/96/T--DTU-Denmark--PromEx.svg" alt="This figure shows a bacterial promoter in front of the fluorescent protein mCherry being replaced by a synthetic promoter when cut with BsaI, thus allowing for expression in our organism." class="safetyfirstimg">		<img style="width: 60%; padding:28px;" src="https://2019.igem.org/wiki/images/9/96/T--DTU-Denmark--PromEx.svg" alt="This figure shows a bacterial promoter in front of the fluorescent protein mCherry being replaced by a synthetic promoter when cut with BsaI, thus allowing for expression in our organism." class="safetyfirstimg">
−	<figcaption>The figure shows the general strategy for replacing the prokaryotic promoter BBa_K3046017 upstream of mCherry with a synthetic promoter using Golden Gate assembly.</figcaption>	+	<figcaption>Figure 1: The figure shows the general strategy for replacing the prokaryotic promoter BBa_K3046017 upstream of mCherry with a synthetic promoter using Golden Gate assembly.</figcaption>
		+	</figure>
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		+	<br>
		+	The use of this device is shown in Figure 2.<br>
		+	When the bacterial promoter is present in the device, mCherry is expressed in <i>E. coli</i> and not in the target eukaryote, such as <i>Aspergillus niger</i>. When the promoter is exchanged, mCherry is no longer expressed in <i>E. coli</i>, but fluorescence can be observed in the eukaryote.
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		+	<figure>
		+	<img style="width: 60%; padding:28px;" src="https://static.igem.org/mediawiki/parts/4/49/T--DTU-Denmark--part9_proof.png" class="safetyfirstimg">
		+	<figcaption>Figure 2: The figure two plates with <i>E. coli</i> colonies, with the ones on the left containing the bacterial promoter and the right one containing a synthetic fungal promoter. Note that few red colonies can be seen on the plate on the right, which serves as a quick screening method for correct insertion of the promoter.</figcaption>
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Revision as of 01:17, 22 October 2019

Fungal MoClo promoter test device

This is a device for testing promoters by expressing mCherry.

Usage and Biology

The bacterial promoter, BBa_K3046017, can be cut out using the Type IIs restriction enzyme as shown in the figure below. Following this, a promoter following the level 0 promoter + 5' UTR MoClo standard can be inserted and the assembled construct can be used for characterization of the promoter.

The use of this device is shown in Figure 2.
When the bacterial promoter is present in the device, mCherry is expressed in E. coli and not in the target eukaryote, such as Aspergillus niger. When the promoter is exchanged, mCherry is no longer expressed in E. coli, but fluorescence can be observed in the eukaryote.

Sequence and Features