"
Team:Cambridge-JIC/Guide/Constructs/Reporters
From 2014.igem.org
(→Fluorescent proteins) |
|||
| (One intermediate revision not shown) | |||
| Line 4: | Line 4: | ||
[http://parts.igem.org/Part:BBa_K165005 Venus YFP], MRFP1 and eGFP. | [http://parts.igem.org/Part:BBa_K165005 Venus YFP], MRFP1 and eGFP. | ||
| - | If you have no choice but to use fluorescent proteins with overlapping spectra, consider using one of the | + | If you have no choice but to use fluorescent proteins with overlapping spectra, consider using one of the localisation tags |
Marchantia also has considerable autofluorescence when stressed. Be sure to use a control when analysing fluorescent constructs to ensure that what you observe is indeed what you were looking for. | Marchantia also has considerable autofluorescence when stressed. Be sure to use a control when analysing fluorescent constructs to ensure that what you observe is indeed what you were looking for. | ||
| Line 17: | Line 17: | ||
LTI: | LTI: | ||
| - | + | [[File:CAM14_eGFP-LTI.png|thumb|center|300px|Fluorescent dissecting scope. Excitation:UV, Leica filter set GFP1]] | |
N7: | N7: | ||
[[File:CAM14_eGFP-N7.png|thumb|center|300px|Confocal Microscopy image of eGFP n7. Excitation:488, Detection range: 520-540.]] | [[File:CAM14_eGFP-N7.png|thumb|center|300px|Confocal Microscopy image of eGFP n7. Excitation:488, Detection range: 520-540.]] | ||
Latest revision as of 15:01, 17 October 2014
Contents |
Reporters
Fluorescent proteins
The following fluorescent proteins have been used with consistent results in Marchantia: [http://parts.igem.org/Part:BBa_K165005 Venus YFP], MRFP1 and eGFP.
If you have no choice but to use fluorescent proteins with overlapping spectra, consider using one of the localisation tags
Marchantia also has considerable autofluorescence when stressed. Be sure to use a control when analysing fluorescent constructs to ensure that what you observe is indeed what you were looking for.
Localisation Tags
A range of localisation tags can be added to enable to distinguish sources of expression and to visualise individual cells and nuclei. These include: [http://parts.igem.org/Part:BBa_K1484104 N7] and [http://parts.igem.org/Part:BBa_K1484106 LTI].
These can be added instead of the stop codon for the reporters. They are incredibly useful when trying to distinguish individual cells, or different fluorescence channels.
These are clearly visible under fluorescence/confocal microscopes. Sample images include:
LTI:
N7:
Fluorescent transformants are visible first from 3-5 days after transformation.
Other reporters
GUS staining has been successfully used to characterise expression, as seen in in this [http://www.researchgate.net/publication/256611354_Comparison_of_the_MpEF1_and_CaMV35_promoters_for_application_in_Marchantia_polymorpha_overexpression_studies paper]
