|
Our team created new BioBricks for expressing and purifying the Cas13a protein used in our CascAID system and BioBricks for investigation of degradation tags with GFP.
Lwa Cas13a:
-To express the core piece of our project, Cas13a, we cloned the protein sequence - in this set of biobricks for the version of Leptotrichia Wadei- into a psB1C3-backbone and fused it with several elements of our part collection.
By equipping the Cas13a with a T7-promoter, a Tphi-Terminator , N-terminal 6xHis/Twin-strep tag for affinity-chromatography and Sumo tag for increased solubility, we created our favorite Biobrick: BBa_K2323001, customized for expression and purification of Lwa Cas13a.
-Other, more basic versions of this construct we submitted are BBa_K2323000 and BBa_K2323001.
-The results gained by the use of our purified Cas13a enzymes is described in (https://2017.igem.org/Team:Munich/Readouts)
-To independently provide a tool for the removal of the affinity tag for our other Cas13a versions from Lsh and Lbu, we improved BBa_K1319004, submitted by the Aachen team 2014, coding for Tobacco Etch Virus-(TEV) protease by inserting the sequence for an N-termianl 6xHis Tag, creating BBa_K2323002 . This way we were able to express and purify the protease ourselves and could also show that it posesses high cleavage activity (https://2017.igem.org/Team:Munich/Improve).
This BioBrick will be useful for any future iGEM using proteins in a cell-free system.
Degradation Tags:
- We created a library of GFP-containing constructs fused to different degradation tags which are targeted by the ClpP machinery of E.coli. The degradation tags for BBa_K2323003, BBa_K2323003, BBa_K2323006, BBa_K2323007, BBa_K2323008, and BBa_K2323009 were pdt2E, ASV, LAA, LVA, and pdt2B, respectively.
Further, the constructs were put under control of a pTet promoter to make them inducable by aTc.
- After induction of protein expression in cell culure and stopping the translation with Chloramphenicol, the reaction rate of degradation for the different tags was measured (Figure 1).
http://parts.igem.org/File:17-10-30_overview.png (Figure 1)
- Since these tags promote degradation at different rates which are described here and in more detail on the respective registry page (http://parts.igem.org/wiki/index.php?title=Part:BBa_K2323003), this degradation tag library can be used by future teams for creation and fine-tuning of bacterial systems.
|
