Difference between revisions of "Part:BBa K1583100"
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<h3> Fluorescence assay </h3> | <h3> Fluorescence assay </h3> | ||
<p> | <p> | ||
− | To be able to ensure that CsgA is expressed, we added a gene encoding for | + | To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (<a href="https://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) under induction of the same rhamnose promoter (<a href="https://parts.igem.org/Part:BBa_K1583112">BBa_K1583112</a>) to check that the promoter works. In this experiment, the fluorescence signal of our csgA construct and csgA-GFP (I13504) constructs was recorded in time after induction with no, 0.2% (w/v) or 0.5% (w/v) rhamnose. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell.All conditions were carried out in triplicates to be able to do a statistical analysis on the data. The different experiments were induced in a 96 well plate. The OD600 and fluorescence signal was recorded in a plate reader during a 18 hour period of induction at 30°C. |
+ | </p> | ||
+ | <p> | ||
In Fig. 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID. | In Fig. 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID. | ||
+ | </p> | ||
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%"> | <img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%"> | ||
<figcaption> | <figcaption> | ||
− | Fig. 1: Fluorescence signal normalized by the number of cells for 0% (w/v), 0.2% (w/v) and 0.5% (w/v) rhamnose with the csgA and csgA-GFPmut3 construct. The error bars are included for all experiments. | + | <b>Fig. 1</b>: Fluorescence signal normalized by the number of cells for 0% (w/v), 0.2% (w/v) and 0.5% (w/v) rhamnose with the csgA and csgA-GFPmut3 construct. The error bars are included for all experiments. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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</p> | </p> | ||
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The calibration line of fluorescence versus mass amount GFPmut3 is given in Fig. 2. | The calibration line of fluorescence versus mass amount GFPmut3 is given in Fig. 2. | ||
+ | </p> | ||
+ | |||
<figure> | <figure> | ||
<img src="https://static.igem.org/mediawiki/2015/9/9e/Modelling_pic_10.png" width="100%" height="100%"> | <img src="https://static.igem.org/mediawiki/2015/9/9e/Modelling_pic_10.png" width="100%" height="100%"> | ||
<figcaption> | <figcaption> | ||
− | Fig. 2: Calibration line of the fluorescence signal of GFPmut3 versus its amount in nanogram. | + | <b>Fig. 2</b>: Calibration line of the fluorescence signal of GFPmut3 versus its amount in nanogram. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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</p> | </p> | ||
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In the modelling, the fluorescent data in Fig. 1 will be further converted to molecules GFPmut3/cell and the promoter activity will be calculated for both the 0.2% (w/v) and 0.5% (w/v) level of rhamnose induction. | In the modelling, the fluorescent data in Fig. 1 will be further converted to molecules GFPmut3/cell and the promoter activity will be calculated for both the 0.2% (w/v) and 0.5% (w/v) level of rhamnose induction. | ||
With this kinetic experiment, we have proven that our csgA-GFPmut3 construct is able to produce different levels of GFPmut3 by varying the rhamnose concentration. | With this kinetic experiment, we have proven that our csgA-GFPmut3 construct is able to produce different levels of GFPmut3 by varying the rhamnose concentration. | ||
+ | </p> | ||
</html> | </html> | ||
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<partinfo>BBa_K1583100 parameters</partinfo> | <partinfo>BBa_K1583100 parameters</partinfo> | ||
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<h3>Reference</h3> | <h3>Reference</h3> | ||
<p> | <p> | ||
“Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, BMC Biotechnology 2008, 8:2 | “Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, BMC Biotechnology 2008, 8:2 | ||
</p> | </p> |
Revision as of 00:28, 18 September 2015
pRha + CsgA
This part is meant to express the ''csgA'' gene under control of L-rhamnose-inducible promoter. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG. This part was designed to be able to control the nanowire formation.
Characterization
This part was characterized in three different experiments:
Fluorescence assay
To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (BBa_I13504) under induction of the same rhamnose promoter (BBa_K1583112) to check that the promoter works. In this experiment, the fluorescence signal of our csgA construct and csgA-GFP (I13504) constructs was recorded in time after induction with no, 0.2% (w/v) or 0.5% (w/v) rhamnose. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell.All conditions were carried out in triplicates to be able to do a statistical analysis on the data. The different experiments were induced in a 96 well plate. The OD600 and fluorescence signal was recorded in a plate reader during a 18 hour period of induction at 30°C.
In Fig. 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID.
![](https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png)
As can be seen from Fig. 1, only the experiments with 0.2% (w/v) and 0.5% (w/v) rhamnose induction with the csgA-GFPmut3 construct gave a clear increase in fluorescence signal in time. All other experiments, gave similar levels of fluorescence, slightly increasing in time. Furthermore, it can be seen that a higher induction level of rhamnose leads to an increase in GFPmut3 and thus fluorescence. Finally, as the fluorescence signal is normalized by the cell density, one can make statements about the activity of the rhamnose promoter. The promoter seems to not be active right after induction, but more after 3 or 4 hours. This is in accordance with data from literature (Wegerer et. al), in which a low amount of fluorescence with a rhamnose promoter was observed after 2 hours of induction.
The calibration line of fluorescence versus mass amount GFPmut3 is given in Fig. 2.
![](https://static.igem.org/mediawiki/2015/9/9e/Modelling_pic_10.png)
The corresponding function of the GFPmut3 calibration line is:
In the modelling, the fluorescent data in Fig. 1 will be further converted to molecules GFPmut3/cell and the promoter activity will be calculated for both the 0.2% (w/v) and 0.5% (w/v) level of rhamnose induction. With this kinetic experiment, we have proven that our csgA-GFPmut3 construct is able to produce different levels of GFPmut3 by varying the rhamnose concentration.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
“Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, BMC Biotechnology 2008, 8:2