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Team:KULeuven/Data/GFP
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K145015 Parts Registry:K145015] | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145015 Parts Registry:K145015] | ||
| + | |||
| + | ===Introduction=== | ||
| + | ''Green fluorescent protein'' (GFP) is often used as a reporter protein, because it allows easy and nondestructive in situ monitoring of cellular processes. It can be expressed in a wide range of organisms and it doesn’t require the addition of a special substrate in order to detect green fluorescence. Nevertheless, the protein has one major drawback, it seems to be very stable. Once the expression of GFP is started, it will fluoresce for a very long period of time. This makes GFP unsuitable for monitoring rapid changes in gene expression. However, in our project fast changes of input will lead to fast changes of output. It is therefore of the utmost importance that we can easily monitor these changes. Andersen et al. [https://2008.igem.org/Team:KULeuven/Data/GFP#References [1<html>]</html>] had already indicated that proper tagging of GFP will make the protein less stable. These researchers showed that a so-called LVA tag seemed to be the most efficient tag to make GFP unstable. This tag consists of a short peptide sequence (AANDENYALVA) and is attached to the C-terminal end of GFP. It is believed that the LVA tag renders GFP susceptible to the action of tail-specific proteases, namely Tsp protease in the periplasm and ClpXP and ClpAP in the cytoplasm of ''E.coli'' [https://2008.igem.org/Team:KULeuven/Data/GFP#References [2<html>]</html>]. | ||
| + | |||
| + | Since we have to monitor the variability of the gene expressions in our system, we decided to employ this fast degradable GFP mutant. We therefore transformed GFP with LVA tag into a BioBrick and characterized it with ''fluorescence-activated cell sorting'' (FACS). | ||
===Materials and methods=== | ===Materials and methods=== | ||
Electrocompetent Top10 cells were electroporated with the | Electrocompetent Top10 cells were electroporated with the | ||
| - | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145015 K145015] part in a pSB1A2 vector. These were plated out on LB plates with ampicillin and grown overnight (37°). From this, a liquid culture supplied with 100 µg ampicillin / ml was prepared and also grown overnight (37°C). These cells were inoculated in fresh LB medium (100 µg ampicillin / ml) in the morning and grown at 37°C for 3.5h. They were then washed in preheated (37°C) ABT minimal medium and resuspended in the same medium and the same amount of medium. After that, cells were | + | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K145015 K145015] part in a pSB1A2 vector. These were plated out on LB plates with ampicillin and grown overnight (37°). From this, a liquid culture supplied with 100 µg ampicillin / ml was prepared and also grown overnight (37°C). These cells were inoculated in fresh LB medium (100 µg ampicillin / ml) in the morning and grown at 37°C for 3.5h. They were then washed in preheated (37°C) ABT minimal medium and resuspended in the same medium and the same amount of medium. After that, cells were whithdrawn at different times and FACS analysis on distribution of fluorescence was done. |
| + | |||
| + | ===Results and discussion=== | ||
| + | under construction... | ||
| + | |||
| + | ===References=== | ||
| + | {| | ||
| + | |-style="vertical-align:top;" | ||
| + | |width=30px| [1] | ||
| + | |height=50px|J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” ''Applied and Environmental Microbiology'', vol. 64, Jun. 1998, pp. 2240–2246. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=9603842 article] | ||
| + | |-style="vertical-align:top;" | ||
| + | |width=30px|[2] | ||
| + | |height=50px|A.W. Karzai et al., "The SsrA−SmpB system for protein tagging, directed degradation and ribosome rescue", ''Nature Structural Biology'', vol. 7, 2000, pp. 449 - 455. [http://www.nature.com/nsmb/journal/v7/n6/full/nsb0600_449.html article] | ||
| + | |} | ||
Revision as of 21:57, 22 September 2008
Contents |
GFP with LVA-tag
Introduction
Green fluorescent protein (GFP) is often used as a reporter protein, because it allows easy and nondestructive in situ monitoring of cellular processes. It can be expressed in a wide range of organisms and it doesn’t require the addition of a special substrate in order to detect green fluorescence. Nevertheless, the protein has one major drawback, it seems to be very stable. Once the expression of GFP is started, it will fluoresce for a very long period of time. This makes GFP unsuitable for monitoring rapid changes in gene expression. However, in our project fast changes of input will lead to fast changes of output. It is therefore of the utmost importance that we can easily monitor these changes. Andersen et al. [1] had already indicated that proper tagging of GFP will make the protein less stable. These researchers showed that a so-called LVA tag seemed to be the most efficient tag to make GFP unstable. This tag consists of a short peptide sequence (AANDENYALVA) and is attached to the C-terminal end of GFP. It is believed that the LVA tag renders GFP susceptible to the action of tail-specific proteases, namely Tsp protease in the periplasm and ClpXP and ClpAP in the cytoplasm of E.coli [2].
Since we have to monitor the variability of the gene expressions in our system, we decided to employ this fast degradable GFP mutant. We therefore transformed GFP with LVA tag into a BioBrick and characterized it with fluorescence-activated cell sorting (FACS).
Materials and methods
Electrocompetent Top10 cells were electroporated with the K145015 part in a pSB1A2 vector. These were plated out on LB plates with ampicillin and grown overnight (37°). From this, a liquid culture supplied with 100 µg ampicillin / ml was prepared and also grown overnight (37°C). These cells were inoculated in fresh LB medium (100 µg ampicillin / ml) in the morning and grown at 37°C for 3.5h. They were then washed in preheated (37°C) ABT minimal medium and resuspended in the same medium and the same amount of medium. After that, cells were whithdrawn at different times and FACS analysis on distribution of fluorescence was done.
Results and discussion
under construction...
References
| [1] | J.B. Andersen et al., “New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria,” Applied and Environmental Microbiology, vol. 64, Jun. 1998, pp. 2240–2246. article |
| [2] | A.W. Karzai et al., "The SsrA−SmpB system for protein tagging, directed degradation and ribosome rescue", Nature Structural Biology, vol. 7, 2000, pp. 449 - 455. article |
