Welcome to the Wiki of Gothenburgs team for iGEM 2014!

This whole page is under very experimental construction.
In a not too distant future you will find here all the amazing things we've learned and achieved.

Click here to edit the home page!

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.

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.

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

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

Requirements

Please be sure to keep these links, your audience will want to find your:

• Home
• Team
• Official Team Profile
• Project
• Parts
• Modeling
• Notebook
• Safety
• Attributions

There are a few wiki requirements teams must follow:

• All pages, images and files must be hosted on the 2014.igem.org server.
• All pages must be created under the team’s name space.
• As part of your documentation, keep the links from the menu to the left.
• Do not use flash in wiki code.
• The iGEM logo should be placed on the upper part of every page and should link to 2014.igem.org.

Visit the Wiki How To page for a complete list of requirements, tips and other useful information.

Tips

We are currently working on providing teams with some easy to use design templates.
In the meantime you can also view other team wikis for inspiration! Here are some very good examples

• 2013 SDU Denmark
• 2013 SYSU China
• 2013 Shenxhen BGIG ATCG
• 2013 Colombia Unianades
• 2013 Lethbridge

For a full wiki list, you can visit iGEM 2013 web sites and iGEM 2012 web sites lists.

This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started:

• State your accomplishments! Tell people what you have achieved from the start.
• Be clear about what you are doing and what you plan to do.
• You have a global audience! Consider the different backgrounds that your users come from.
• Make sure information is easy to find; nothing should be more than 3 clicks away.
• Avoid using very small fonts and low contrast colors; information should be easy to read.
• Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the iGEM 2013 calendar
• Have lots of fun!