Team:BOKU-Vienna/Basic Part

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Project Inspiration Description

Basic Parts

Mycolactone-Riboswitch BBa_K3015000

We successfully screened for a riboswitch that will increase transcription of a downstream gene. This is the centerpiece of our collection, since this is the Part that could make the detection of Mycolactone easier than ever.
It Consists of an Aptamer-sequence specific to Mycolactone, a spacer region, a terminator-sequence and an U-Stretch. In the absence of Mycolactone the terminator-sequence will bind to the aptamer-sequence and form a hairpin structure, which will bind to the RNA-Polymerase and force it to dissociate from the DNA-strand. In the presence of Mycolactone it will bind to the Aptamer-sequence and prevent the hairpin structure from forming and the RNA-Polymerase can transcribe. Mycolactone-Riboswitch

RNA secondary structure prediction [1]

Theophylline-Riboswitch BBa_K3015004

The iGEM Team BOKU-Vienna improved the Part BBa_K784005 by designing a Theophylline riboswitch that works in a transcriptional way, instead of a translational way. The coding sequence does not have to be scarless fused to the riboswitch, which makes cloning easier. To test the probabilities of the riboswitch we conducted measurements by fusing the riboswitch downstream to a Promoter and upstream to an RBS and GFP. This is important for the riboswitche´s function because the Ribosome needs to dissociate before the RBS can be transcribed. You can find those measurements on the Regsitry-Page of the described Composite Part BBa_K3015007 Theophyllin-Riboswitch

RNA secondary structure prediction[2]

T7-Promoter LacOperator RBS BBa_K3015012

This T7-Promoter with a Lac Operator was used to drive the expression of our blue Chromoprotein [2]
Note: The Lac-Operator had no function in our system, because there was no LacI-gene and therefore no repressor involved. We only made use of the T7-Promoter and RBS sequence. But we still included the Lac-Operator since it makes the part more applicable.

T7 RNA Polymerase (No BbsI/BpiI) BBa_ K3015006

Since BbsI/BpiI can be a very important restriction enzyme for golden gate cloning in some labs, we decided to remove those recognition sites by codon swapping from the Part BBa_K14001 to improve the applicability

GFP-mut3 BBa_K3015013

GFPmut3 is an optimized variant of the GFP from Aequorea victoria with the following substitutions:
S65G
S72A
Those substitutions will improved folding and excitation at 488nm [3]

Referenzen

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