Team:Munich/Parts


Parts

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_K2323004, 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 readouts.

TEV protease

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 with this improved BioBrick.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_K2323006, BBa_K232300, 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).

Figure 1: First order kinetics of degradation for different protein-degradation tags.

Since these tags promote degradation at different rates which are described here and in more detail on the respective registry page, this degradation tag library can be used by future teams for creation and fine-tuning of bacterial systems.

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