Difference between revisions of "Part:BBa K3046009"
 
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===Usage and Biology===
 
===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 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.  
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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">
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<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>
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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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We have demonstrated the use of this device in Figure 2 where a promoter from the <a href="https://2019.igem.org/Team:DTU-Denmark/Part_Collection" target="_blank">LEAP library</a> has been inserted.<br>
  
 
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<img style="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">
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<img style="width: 60%; padding:28px;" src="https://static.igem.org/mediawiki/parts/4/49/T--DTU-Denmark--part9_proof.png" class="safetyfirstimg">
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<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>
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<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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Latest revision as of 01:33, 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.

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.
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.


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. We have demonstrated the use of this device in Figure 2 where a promoter from the LEAP library has been inserted.
Figure 2: The figure two plates with E. coli 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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1158
    Illegal BamHI site found at 1418
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 232
    Illegal BsaI.rc site found at 6