Team:TU-Eindhoven/LEC/LabTheory

• Introduction
  • Home page
  • Background
    information
  • About us
  • Contact us
  • Our photos
• Project
  • Project description
  • Lab approach
  • Device information
  • Initial model
  • Yeast model
  • Lab results
  • Our BioBricks^TM
  • Our
    accomplishments
• Considerations
  • Public outreach
  • Safety questions
  • Other thoughts
• Future applications
  • Human interest
  • Further
    developments
• Notebook
  • Before week 1
  • Week 1
  • Week 2
  • Week 3
  • Week 4
  • Week 5
  • Week 6
  • Week 7
  • Week 8
  • Week 9
  • Week 10
  • Week 11
  • Week 12
  • After week 12
  • Lab protocols
• Partners
  • ICMS TU/e
  • Team collaborations
  • Attributions and Contributions
  • Our sponsors

Since, the aim of this project is to design and produce a new multi-color display in which genetically engineered cells function as pixels analogous to how a flat panel display works and the decision has been made that yeast cells are, in this case, the most practical to work with, concerning multiple reasons discussed in section ‘Yeast versus E. Coli’, light emitting yeast cells which are sensitive to electric stimuli have to be engineered and produced. Considering this goal, the lab-team faces the challenge of introducing sensors sensitive to electrical stimuli resulting in an emission of light. After extensive literature research, it is hypothesized that CCH1-MID1 calcium plasma membrane channels, found in Saccharomyces cerevisiae but homologous to mammalian voltage-gated calcium channels, are able to facilitate a calcium influx upon plasma membrane depolarization (<a href="#ref_Iida" name="text_Iida">^[1]</a>). Furthermore, it is known that GECO proteins (<a href="#ref_zhao" name="text_zhao">^[2]</a>) are sensitive to calcium resulting in fluorescence upon increased concentration. Regarding both, hypothesis and known fact, laboratory work could start.

DNA coding for CCH1, MID1 and the three colours (red, green, blue) GECO protein are obtained from H. Iida & K. Iida and Zhao, Campbell group via Addgene respectively. Via PCR these DNA strengths are ligated in vectors pBCT, YCpT and pYES3 respectively. Then, vectors coding for CCH1, MID1 and one of the three GECO colours were transformed into INVSc1 S. Cerevisiae cells using a heat shock protocol (more information can be found in section ‘Protocols’), obtaining three different strains of yeast, all containing the CCH1-MID1 calcium channel but each with a different colour GECO.

The genetically modified S. Cerevisiae cells were put to the test in our home-made device, designed for providing electrical stimuli to the yeast cells. More about the device can be found in section ‘Device information’. These tests are done in a single bath filled with SC media containing nutrients for our genetically modified yeast and calcium. After electrical stimulation of yeast at different positions on the device, optical signals are seen by the naked eye and characterized by a plate reader.

Furthermore, a BioBrick^TM of the GECO protein was designed and prepared using the coding DNA from Zhao and restriction enzymes. The obtained a BioBrick^TM was ligated into a pET28a vector and transformed into BL21 competent cells. Using IPTG, the GECO proteins were expressed after which there properties were characterized. More about the design, preparation, expression and characterization can be read in section ‘BioBrick^TM’.

Off Course, there will always be problems, struggles and obstacles which have to be overcome during laboratory work. Section ‘Struggles and Solutions’ more can be read about the struggles our team had to face during our project and the steps we took to overcome these obstacles.

References

• [1] Iida, K. et al. 2007, 10.1074/jbc.M703757200
• [2] Zhao et al. 2011, 10.1126/science.1208592

Yeast versus E. Coli

Before this light emitting cell display project could start, it was necessary to decide whether E. Coli or yeast would be used. As both competent cell types are able to develop rapidly, take care of protein expression and are cheap to culture, the decision was made upon the difference in complexity of the cell types. Not only with a few native calcium channels, which exist in both cell types, our iGEM team would be able to reach the goal of this project, but also overexpression of voltage-gated calcium channel was needed. As soon as it was found that CCH1-MID1, a homologous to mammalian voltage-gated calcium channels, could be overexpressed in S. Cerevisiae and facilitates the influx of calcium upon electrical stimuli, it was easily decided to use yeast as competent cells for our project.

GECOs

Real-time imaging of biochemical events inside living cells is important for understanding the molecular basis of physiological processes and diseases <a href="#ref_merkx” name=”text_merkx”>^[1]</a>. Genetically encoded sensors based on fluorescent proteins (FPs) are frequently used for molecular recognition. In this iGEM project we use the fluorescent proteins for providing the light in our display.

A GECO is a protein which emits light in the presence of Ca²⁺<a href="#ref_zhao” name=”text_zhao”>^[2]</a>. There are two important classes of genetically encoded Ca²⁺ indicators. One is called the Forster Resonance Energy Transfer (FRET)-based cameleon type<a href="#ref_miyawaki” name=”text_miyawaki”>^[3]</a> and the other one is called the single Green Fluorescent Protein (GFP) type<a href="#ref_nakai” name=”text_nakai”>^[4]</a>. The GECO protein belongs to the single GFP type. Research has shown that Ca²⁺ indicators targeted to the E.coli periplasm can be shifted toward the Ca²⁺-free of Ca²⁺ -bound states by manipulation of the environmental Ca²⁺ concentration<a href="#ref_zhao” name=”text_zhao”>^[2]</a>. Robert E. Campbell et al. named those Ca²⁺ indicators GECO’s. R-GECO, G-GECO and B-GECO emit respectively red, green or blue light with each another emission and excitation spectra (Fig. 1 and Fig2).

The GECO has been implemented into the DNA of the yeast cells with the help of a YES3/CT plasmid (Fig. 3). After transcription and translation the protein emits light if there is enough Ca²⁺ in the cytoplasm of the yeastcell. Light emission can only be established if the Ca²⁺ threshold in the cytoplasm is exceeded. References

• [1] Laurens Lindenburg and Maarten Merkx, ‘Colorful Calcium Sensors’, 2012
• [2] Robert E. Campbell et al., ‘An Expanded Palette of Genetically Encoded Ca²⁺ Indicators’, 2011
• [3] A. Miyawaki et al., Nature 338, 1997
• [4] J. Nakai, M. Ohkura, K. Imoto, Nat. Biotechnol. (2001)