Team:Gothenburg

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<b>Woopwoop!</b>
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<p><a href="https://2014.igem.org/Team:Gothenburg/SampleTemplate"/a> Template </a></p>
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<p><a href="https://2014.igem.org/Team:Gothenburg/Calendar"/a> Calendar</a></p>
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<h3>Introduction</h3>
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<h2>Introduction</h2>
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<p>Our project intends to create a generation counter in Saccharomyces cerevisiae. The yeast cell would express fluorescent proteins of different colors according to the number of daughter cells spawn. Nowadays, the replicative age of a yeast cell is determined by counting the budding scars with the help of a microscope. This process is time-consuming and not automated. Once our idea is implemented, it would be possible to use a flow cytometric device to sort the cells according to their color and, consequently, generation number. To achieve that, we are building genetic circuits with a logical AND gate ensuring generation specific expression of different fluorescent proteins.
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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.
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<h3>Introduction</h3>
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<p>Figure 1. Schematic representation of the logical AND gate with the input
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and output signals.
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<h3>Project Description</h3>
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Nowadays the determination of replicative age of yeast cells is done by  
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counting the budding scars of each cell in a microscope, a process time consuming
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and not effective. Our team goal with the iGEM project is to construct a yeast
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generation counter. The idea is that each time the cell divides a different
+
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florescent protein [1, 2] is produced. Therefore by examining the cell under a
+
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microscope or in a flow cytometer one can determine how many times the cell has
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divided. This would be achieved by constructing a logical AND gate in the cell [3]
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where the input signals consist of a cyclin activated dCas9-VP64 and a guide RNA
+
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(gRNA) [4] signal from the previous cell cycle. The output response consist of a
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different fluorescent protein and a new gRNA molecule, see figure 1.
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dCas9-VP64 is an engineered turntable transcription factor only active when dimerized
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with an interchangeable gRNA molecule. The gRNA also determines the specificity of
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the transcription factor which enables the dCas9-VP64 to activate different genes
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depending on the sequence of the gRNA molecule and the promoter. gRNA consists of two
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parts, a scaffold and a 20 bp Specificity Determinant Sequence (SDS) on the 5' end.
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The scaffold constitutes the majority of the gRNA molecule and gives it its structure
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whereas the SDS binds to the target site in the gene promoter.  Cyclins are proteins
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that are involved in the progression of the cell cycle, therefore activated at specific
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times [5]. To mimic the specific production pattern of cyclins the dCas9-VP64 gene is
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placed under the control of a yeast cyclin promoter. Therefore the Cas9 will be
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produced in the G1 phase. When the Cas9 and gRNA dimerize and the transcription factor
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is activated, it in turn activates the transcription of a new fluorescent protein and
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a new gRNA molecule to act as a memory for the next cycle. Once this age counter is
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implemented, it would be possible to sort cells according to their replicative age
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automatically with a flow cytometer device.
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</p>
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<h4>References</h4>
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<ol id="references">
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<li>Hackett, E.A., et al., A family of destabilized cyan fluorescent proteins as
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transcriptional reporters in S. cerevisiae. Yeast, 2006. 23(5): p. 333-349.</li>
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<li>Andersen, J.B., et al., New unstable variants of green fluorescent protein for
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studies of transient gene expression in bacteria. Applied and environmental microbiology,
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1998. 64(6): p. 2240-2246.</li>
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<li>Moon, T.S., et al., Genetic programs constructed from layered logic gates in single
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cells. Nature, 2012. 491(7423): p. 249-253.</li>
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<li>Farzadfard, F., S.D. Perli, and T.K. Lu, Tunable and multifunctional eukaryotic
 
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transcription factors based on CRISPR/Cas. ACS synthetic biology, 2013. 2(10): p.
 
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604-613.</li>
 
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<li>Nasmyth, K., At the heart of the budding yeast cell cycle. Trends in Genetics,
 
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1996. 12(10): p. 405-412</li>
 
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Latest revision as of 12:15, 17 October 2014

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Introduction

Our project intends to create a generation counter in Saccharomyces cerevisiae. The yeast cell would express fluorescent proteins of different colors according to the number of daughter cells spawn. Nowadays, the replicative age of a yeast cell is determined by counting the budding scars with the help of a microscope. This process is time-consuming and not automated. Once our idea is implemented, it would be possible to use a flow cytometric device to sort the cells according to their color and, consequently, generation number. To achieve that, we are building genetic circuits with a logical AND gate ensuring generation specific expression of different fluorescent proteins.


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