Team:Gothenburg

Introduction

Nowadays the determination of replicative age of yeast cells is done by counting the budding scars of each cell in a microscope, a process time consuming and not effective. Our team goal within the iGEM competition is to construct a yeast generation counter. The idea is that each time the cell divides a different florescent protein [1, 2] is produced. Therefore by examining the cell under a microscope or in a flow cytometer one can determine how many times the cell has divided. This would be achieved by constructing a logical AND gate in the cell [3] where the input signals consist of a cyclin activated dCas9-VP64 and a guide RNA (gRNA) [4] signal from the previous cell cycle. The output response consist of a different fluorescent protein and a new gRNA molecule, see figure 1.

Introduction

Figure 1. Schematic representation of the logical AND gate with the input and output signals.

Project Description

Nowadays the determination of replicative age of yeast cells is done by counting the budding scars of each cell in a microscope, a process time consuming and not effective. Our team goal with the iGEM project is to construct a yeast generation counter. The idea is that each time the cell divides a different florescent protein [1, 2] is produced. Therefore by examining the cell under a microscope or in a flow cytometer one can determine how many times the cell has divided. This would be achieved by constructing a logical AND gate in the cell [3] where the input signals consist of a cyclin activated dCas9-VP64 and a guide RNA (gRNA) [4] signal from the previous cell cycle. The output response consist of a different fluorescent protein and a new gRNA molecule, see figure 1.

dCas9-VP64 is an engineered turntable transcription factor only active when dimerized with an interchangeable gRNA molecule. The gRNA also determines the specificity of the transcription factor which enables the dCas9-VP64 to activate different genes depending on the sequence of the gRNA molecule and the promoter. gRNA consists of two parts, a scaffold and a 20 bp Specificity Determinant Sequence (SDS) on the 5' end. The scaffold constitutes the majority of the gRNA molecule and gives it its structure whereas the SDS binds to the target site in the gene promoter. Cyclins are proteins that are involved in the progression of the cell cycle, therefore activated at specific times [5]. To mimic the specific production pattern of cyclins the dCas9-VP64 gene is placed under the control of a yeast cyclin promoter. Therefore the Cas9 will be produced in the G1 phase. When the Cas9 and gRNA dimerize and the transcription factor is activated, it in turn activates the transcription of a new fluorescent protein and a new gRNA molecule to act as a memory for the next cycle. Once this age counter is implemented, it would be possible to sort cells according to their replicative age automatically with a flow cytometer device.

References

1. Hackett, E.A., et al., A family of destabilized cyan fluorescent proteins as transcriptional reporters in S. cerevisiae. Yeast, 2006. 23(5): p. 333-349.
2. Andersen, J.B., et al., New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Applied and environmental microbiology, 1998. 64(6): p. 2240-2246.
3. Moon, T.S., et al., Genetic programs constructed from layered logic gates in single cells. Nature, 2012. 491(7423): p. 249-253.
4. Farzadfard, F., S.D. Perli, and T.K. Lu, Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS synthetic biology, 2013. 2(10): p. 604-613.
5. Nasmyth, K., At the heart of the budding yeast cell cycle. Trends in Genetics, 1996. 12(10): p. 405-412