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Team:Cambridge-JIC/Constructs/progress template
From 2014.igem.org
(Difference between revisions)
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<h4> Aim of the construct </h4> | <h4> Aim of the construct </h4> | ||
<ul> | <ul> | ||
| - | + | Chromoproteins represent ideal reporter genes. Unlike fluorescent protein, from which chromoproteins were originally derived, they require no specialised optical equipment for detection. Our aim is to use a selection of such proteins, donated by the iGEM 2012 Uppsala team, as our most basic and main output in regards to the Marchantia framework. | |
| - | <br> | + | <br></br> |
We decided to test the following chromoprotein constructs: | We decided to test the following chromoprotein constructs: | ||
| - | <br> | + | <br></br> |
<li> 35s - eforRed - nosT</li> | <li> 35s - eforRed - nosT</li> | ||
<li> 35s - eforRed - N7 - nosT</li> | <li> 35s - eforRed - N7 - nosT</li> | ||
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<li> 35s - asPink - N7 - nosT</li> | <li> 35s - asPink - N7 - nosT</li> | ||
<li> 35s - aeBlue - N7 - nosT</li> | <li> 35s - aeBlue - N7 - nosT</li> | ||
| - | <br> | + | <br></br> |
By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis. | By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis. | ||
The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye. | The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye. | ||
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</ul> | </ul> | ||
Revision as of 14:11, 24 July 2014
Chromoprotein Constructs
Design
Aim of the construct
-
Chromoproteins represent ideal reporter genes. Unlike fluorescent protein, from which chromoproteins were originally derived, they require no specialised optical equipment for detection. Our aim is to use a selection of such proteins, donated by the iGEM 2012 Uppsala team, as our most basic and main output in regards to the Marchantia framework.
- 35s - eforRed - nosT
- 35s - eforRed - N7 - nosT
- 35s - amilCP - N7 - nosT
- 35s - tsPurple - nosT
- 35s - tsPurple - N7 - nosT
- 35s - asPink - N7 - nosT
- 35s - aeBlue - N7 - nosT
We decided to test the following chromoprotein constructs:
By expressing the chromoproteins in the pGreen vector with the 35s promoter and the nosT terminaor, we control for the possiblity of the promoter/terminator not functioning in our chassis. The N7 fragment allows for the selected constructs to localise chromoprotein to the nucleus. It is believed by Bernardo Pollak that this may end up increasing the likelihood of detecting the chromoproteins with the naked eye.
End goal plasmid
- insert image here
Experimentation (up to date)
PCR
Attempt 1:- Start date/time:
- End date/time:
- UIDs of primers/plasmids used:
- A2, P12, P13
- B3, P14, P13
- A4, P12, P13
- PCR Spreadsheet (on google drive)
- Gel 27/07/2014
Gibson
Attempt 1:- Start date/time:
- End date/time:
- Comments:
E-Coli transformation
Attempt 1:- Start date/time:
- End date/time:
- Comments:
Agrobacteria transformation
Attempt 1:- Start date/time:
- End date/time:
- Induce comments:
- Growth comments:
- Electroporation/selection comments:
Spore preparation
Attempt 1:- Start date/time:
- End date/time:
- Comments:
Spore transformation
Attempt 1:- Start date/time:
- End date/time:
- Comments:
Evaluation
- Start date/time:
- End date/time:
- Comments:
