Difference between revisions of "Part:BBa K243032"
(→Purification) |
(→Detection) |
||
| (8 intermediate revisions by the same user not shown) | |||
| Line 3: | Line 3: | ||
| − | This part is | + | This part is composed of the essential Fok_a part [https://parts.igem.org/Part:BBa_K243000 protein domain Fok_a] of our universal endonuclease, but additionally linked to a [https://parts.igem.org/Part:BBa_K157008 Venus-marker] to detect even low levels of protein expression by fluorescence microscopy. |
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | The addition of a fluorescent protein(Venus) to the protein construct enables | + | The addition of a fluorescent protein (Venus) to the protein construct enables easy detection of Fok_a expression by fluorescence microscopy as well as a new way of purification by a GFP-trap column. The Venus protein was C-terminally fused to the Fok_a protein domain. In the opposite orientation, skipped expression of Fok_a would not be detectable. When the fusion protein is expressed by E. coli, the fluorescence signal of the Venus protein can be detected. In addition to these methods, anti-GFP antibodies can be used to check both the protein size and presence by western blotting. |
<br> | <br> | ||
===Detection=== | ===Detection=== | ||
| − | + | A strong fluorescence signal could be detected using the fluorescence microscope and IPTG-induced cells. ''E. coli''(RV308) were excited by light of the wavelength 505 nm and fluorescence pictures were made using a cutoff filter.<br> | |
| − | [[Image: | + | [[Image:FokA Venus.JPG|500x500px]]<br> |
The cell extracts were checked by SDS-page to get information about the expression of proteins.<br><br> | The cell extracts were checked by SDS-page to get information about the expression of proteins.<br><br> | ||
| Line 21: | Line 21: | ||
SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced) | SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced) | ||
---- | ---- | ||
| − | In the lanes of the induced clones ( lanes 3,4) a band of about 50 kDA appears which could | + | In the lanes of the induced clones ( lanes 3,4) a band of about 50 kDA appears which could represent the Fok_a_Venus protein.<br><br> |
| − | A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP)was conducted to prove | + | A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP) was conducted to prove expression of the complete Fok_a_YFP construct although many Fok_a constructs were hard to express.<br><br> |
[[Image:Freiburg09 WBfokvenus.jpg]]<br> | [[Image:Freiburg09 WBfokvenus.jpg]]<br> | ||
---- | ---- | ||
| Line 28: | Line 28: | ||
---- | ---- | ||
<br> | <br> | ||
| − | All of the induced clones show a strong signal but also many different bands on the membrane. There is a signal | + | All of the induced clones show a strong signal but also many different bands on the membrane. There is a signal corresponding to 49 kDa which matches the theoretical size of the Fok_a_Venus fusion protein. The bands indicating proteins of smaller size are assumed to be degradation products. |
<br><br> | <br><br> | ||
Latest revision as of 04:07, 22 October 2009
Fok_a-Venus
This part is composed of the essential Fok_a part protein domain Fok_a of our universal endonuclease, but additionally linked to a Venus-marker to detect even low levels of protein expression by fluorescence microscopy.
Usage and Biology
The addition of a fluorescent protein (Venus) to the protein construct enables easy detection of Fok_a expression by fluorescence microscopy as well as a new way of purification by a GFP-trap column. The Venus protein was C-terminally fused to the Fok_a protein domain. In the opposite orientation, skipped expression of Fok_a would not be detectable. When the fusion protein is expressed by E. coli, the fluorescence signal of the Venus protein can be detected. In addition to these methods, anti-GFP antibodies can be used to check both the protein size and presence by western blotting.
Detection
A strong fluorescence signal could be detected using the fluorescence microscope and IPTG-induced cells. E. coli(RV308) were excited by light of the wavelength 505 nm and fluorescence pictures were made using a cutoff filter.
The cell extracts were checked by SDS-page to get information about the expression of proteins.
SDS gel; pEx Venus-Fok_a in BL21de3; lanes: Marker (ColorPlus Prestained Protein Marker, Broad Range), clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-Dig-SplitLi-Fok_a) uninduced, control induced)
In the lanes of the induced clones ( lanes 3,4) a band of about 50 kDA appears which could represent the Fok_a_Venus protein.
A western blot with specific anti-GFP antibodies (first antibody: Santa Cruz Biotechnology sc-9996 mouse αGFP; second antibody: Santa Cruz Biotechnology sc-2005 goat αmouse IgG-HRP) was conducted to prove expression of the complete Fok_a_YFP construct although many Fok_a constructs were hard to express.
Western Blot; pEx Venus-Fok_a in BL21de3; lanes: Marker (NEB prestained protein marker broad range, clone 1 uninduced, clone 1 induced, clone 1 induced, clone 2 uninduced, clone 2 induced, control (His-FluA-SplitLi-Fok_i + His-DigA-SplitLi-Fok_a) uninduced, control induced)
All of the induced clones show a strong signal but also many different bands on the membrane. There is a signal corresponding to 49 kDa which matches the theoretical size of the Fok_a_Venus fusion protein. The bands indicating proteins of smaller size are assumed to be degradation products.
Purification
It is possible to use the GFP tag or as in our case YFP for purification by a GFP specific column.
Due to the lack of time at the end, the purification of the Fok_a proteins by a GFP column is under progress.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 487