Team:TU-Eindhoven/LEC/Lab

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• 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

Plasmid construction

GECO protein expression and isolation

The three GECOs have been expressed in E. coli BL21 to yield a large amount of the proteins for characterization. Cells were cultured in large Erlenmeyer flasks. The results were visually impressive: After lysis the flasks turned vibrantly red and green respectively. The blue GECO however looked much the same as the green GECO.

TODO: COLUMN FILTRATION

Yeast transformation

A weekly item on our schedule was yeast transformation. It takes about a week to complete a transformation, that is, to add one plasmid to an existing strain variant. Because success is not guaranteed we choose to introduce plasmids in various orders in parallel. This resulted in many variants that we assigned a unique number for convenience. The same number was used on plates, cultures and cryostocks. In total 49 variants were made.

Initially, yeast transformations failed or yielded only several colonies, a lot less than expected. A review of transformation protocols showed that the shock protocol we used should be more efficient than electroporation or other common methods. We tried to transform another yeast strain with our plasmids which yielded the expected high transformation efficiency. Unfortunately that strain was not compatible with the auxotrophic markers on the plasmids we wanted to introduce. Our problem was remedied by using more plasmid DNA for the transformation, as described in our modified yeast transformation protocol.

Device tests

The device was tested with yeast containing all three plasmids, thus over expressing channels and expressing GECOs. A quick inspection with the naked eye did not show any sign of visual light coming off the device, not even in a dark room. We think that the yeast was not concentrated enough to show a clear response.

Spectrophotometry

The following non-BioBrick plasmids have been constructed:

name	vector	inserts	description
pYES2/CT-X-GECO	pYES2/CT	R-GECO1, G-GECO1.1, B-GECO1	High copy-number shuttle vector
pYES3/CT-X-GECO	pYES3/CT	R-GECO1, G-GECO1.1, B-GECO1	High copy-number shuttle vector
pET28a	X-GECO	pET28a	R-GECO1, G-GECO1.1, B-GECO1	Expression vector for E. coli

The following plasmids were received from addgene.org: <thead></thead> <tbody> </tbody>

name	vector	inserts	description
CMV	R-GECO1, G-GECO1.1, B-GECO1	Shipping vector use by addgene.org, CMV vector was not used in this project

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These plasmids were kindly donated by H. Iida & K. Iida:

< td>pBCT-CCH1H-EGFP

name	vector	inserts	description
pBCT -CCH1H	pBCT	CCH1H	Low copy-number shuttle vector, encodes the CCH1 protein
pBCT-CCH1H-HA4	pBCT	CCH1H-HA4	Low copy-number shuttle vector, encodes HA4-tagged CCH1
pBCT	CCH1H-EGFP	Low copy-number shuttle vector, encodes EGFP-tagged CCH1
YCpT-MID1	YCpT	MID1	Low copy-number shuttle vector, encodes the MID1 protein
YCpT-MID1-EGFP	YCpT	MID1-EGFP	Low copy-number shuttle vector, encodes EGFP-tagged MID1

References