Team:Washington/gRNA Expression
Background
Our project was inspired by the work of the Koffas lab¹. Researchers developed a repressible promoter system using sgRNA sequences coupled with the CRISPR/dCas9 mechanism. They incorporated these promoters into the biosynthetic violacein pathway and were able to successfully throttle carbon flux in E. coli, effectively turning promoters into orthogonal on/off switches commanding the direction of metabolic production in bacteria. We realized the boundless implication of this method and thus decided to incorporate this construct into our yeast system.
In order to realize our goal of creating an autonomous yeast culture management platform, we are employing a violacein pathway to visualize metabolic processes in yeast. The pathway consists of five genes: VioA, VioB, and VioE (which are constitutively expressed), VioC and VioD. A single promoter determines expression of VioA, VioB, and VioE, so in our diagrams, the three genes are abbreviated to VioABE. The genes are controlled by inducible promoters. The Dueber Lab performed HPLC on yeast that expressed this pathway to reveal that primarily predeoxyvioacein, deoxyviolacein, proviolacein, and violacein were produced in mass quantities⁴. Culture pigment expression changes according to the following gene activation combinations:
VioABE + VioC → deoxyviolacein
VioABE + VioD → proviolacein
VioABE + VioC + VioD → violacein
Methods
The regulation of our system has several steps:
- sgRNAs that target either VioC or VioD are transcribed depending on how they are expressed. In our design this is based on Cup1 and modGal1 inducible promoters.
- The sgRNAs form a complex with dCas9.
- The large complex will bind to either VioC or VioD and prevent the transcription of these genes into mRNA.
- While VioABE are always transcribed and translated to form predeoxyviolcein, VioC and VioD expression varies based upon the presence of the sgRNA/dCas9 complexes.
- The alternate forms of violacein vary in color. The observed color of a liquid culture of the transgenic yeast changes based on the composition of the forms of violacein produced. Achieving changes in color is possible by changing the expression of VioC and VioD.
The sgRNAs were uniquely designed in yeast to both accurately target VioC and VioD and to contain ribozymes to keep the genes within the nucleus. More specifically, ribozymes were placed upstream and downstream of the sgRNA sequences to “cut” off the 5’ cap and Poly-A tail attached during mRNA processing that marks RNA to leave the nucleus.²
Additionally, we designed the sgRNA plasmid with inducible promoters, modGal1 and CUP1. ModGal1 is coupled with a zinc finger transcription factor that is induced by the presence of beta-estradiol. We included this transcription factor to be constitutively expressed in the same plasmid. CUP1 is a promoter whose transcription factor is induced by the presence of copper to express the VioD sgRNA. Its transcription factor ACE is naturally expressed in the yeast genome.
The sgRNA plasmid was designed to be constructed through Gibson assembly. The insert with the sgRNAs would be ordered, and then assembled with a linearized pRS425 as a vector. This vector contains ampicillin resistance for E. coli selection and leucine for yeast selection.
Problems arose when we first attempted to order the designed insert. Our modified GAL1 promoter with its zinc finger binding site had too many sequence repeats. The team then attempted to use a restriction enzyme (BaeI) to linearize the plasmid. This linearized plasmid would be assembled with a second geneblock. The plasmid carrying the modified GAL1 promoter was never able to be linearized. As a result, we had to switch this promoter for ZAP1. Additionally, the designed insert was too many base pairs to order in one piece at 5kb. This was solved by ordering 2 inserts with homology and first attaching them together through Gibson assembly. Another Gibson assembly was performed to complete the plasmid, and finally, the plasmid was transformed into DH5-alpha E. coli.
We integrated the Violacein pathway,pWCD1133, and pdCas9/Mxi1, pMOD4-CYC1-dCas9-mxi1, into the yeast genome at the URA3 and TRP1 loci respectively. The inducible sgRNAs responsible for guiding the dCas9/Mxi1 complex to its target site were transformed in a non-integrating plasmid. Collectively, this three-plasmid system is intended to create a strain capable of producing all four colors to be analyzed by the Chromastat.
Results
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