Difference between revisions of "Part:BBa K608404"

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	===Usage and Biology===		===Usage and Biology===
	====Experimental setup====		====Experimental setup====
−	According to Adey et al. the plastic binding domain (pbd) binds to the polystyrene surface of micro titer plates (96 well plates). To investigate the binding properties of the plastic binding tag we started several spectroscopic assays using a plate reader (FLUOstar Omega) and polystyrene plates (Greiner bio one) To detect the fluorescence of the GFP tagged to the plastic binding domain we used black plates and a well scanning program measuring 10x10 spots in each well of the micro titer plate. We performed several washing steps to find out how much of the proteins can be found in the eluate and how much remains bound on the plate’s surface. To compare the pbd-tagged GFP to a normal GFP without special plastic binding ability we also measured GFP obtained via expression with our diverse PR (Promoter-Ribosome-binding-site constructs). To affirm the results obtained by fluorescence spectroscopy used the Bradford assay and screened for different protein concentrations in transparent polystyrene plates.	+	According to ''Adey et al.'' the plastic binding domain (pbd) binds to the polystyrene surface of micro titer plates (96 well plates). To investigate the binding properties of the plastic binding tag we started several spectroscopic assays using a plate reader (FLUOstar Omega) and polystyrene plates (Greiner bio one) To detect the fluorescence of the GFP tagged to the plastic binding domain we used black plates and a well scanning program measuring 10x10 spots in each well of the micro titer plate. We performed several washing steps to find out how much of the proteins can be found in the eluate and how much remains bound on the plate’s surface. To compare the pbd-tagged GFP to a normal GFP without special plastic binding ability we also measured GFP obtained via expression with our diverse [http://2011.igem.org/Team:Freiburg/Results#commons PR] (Promoter-Ribosome-binding-site constructs). To affirm the results obtained by fluorescence spectroscopy used the Bradford assay and screened for different protein concentrations in transparent polystyrene plates.
		+	The calibration lines necessary for calculation of protein concentration can be found [[Media:Bradford+Fluorescence calibration.pdf|here]].
	<br/>		<br/>
	====Results====		====Results====
−	After the first measurement of the basic fluorescence intensity we transferred the samples onto another well, refilled the exhausted well with PBS and measured both eluate and remaining protein. In a second washing step the liquid was taken out of the first well again and given to another well. This washing was performed a third time, resulting in three eluates and a triply washed well with more or less protein remaining on the walls.	+	[[Image:picture 1 pbd.jpg|300px|left|thumb|Picture 1: % of tagged (red) or untagged (blue) GFP remaining in the well after washing compared to previous washing step / original concentration]] After the first measurement of the basic fluorescence intensity we transferred the samples onto another well, refilled the exhausted well with PBS and measured both eluate and remaining protein. In a second washing step the liquid was taken out of the first well again and given to another well. This washing was performed a third time, resulting in three eluates and a triply washed well with more or less protein remaining on the walls.
−	[[Image:picture 1 pbd.jpg|300px|left|thumb|Picture 1: % of tagged (red) or untagged (blue) GFP remaining in the well after washing compared to previous washing step / original concentration]]	+
	As shown in picture 1, both pbd-tagged and untagged GFP fluorescence decreases after the first washing step. Only 0- 4% of the original concentrations of the untagged GFP and 2-7% of the pbd-tagged GFP remained in the well. After a second washing step there were differences observable between the tagged and the untagged GFP.  While the percentage of the remaining “normal” GFP was scattering around zero, averagely 60% of the pbd-tagged GFP remained in the well. In a third washing steps this observation was confirmed. While again the main part of the pbd-GFP remained in its original well there could rarely been found any untagged GFP that hadn’t been washed away.		As shown in picture 1, both pbd-tagged and untagged GFP fluorescence decreases after the first washing step. Only 0- 4% of the original concentrations of the untagged GFP and 2-7% of the pbd-tagged GFP remained in the well. After a second washing step there were differences observable between the tagged and the untagged GFP.  While the percentage of the remaining “normal” GFP was scattering around zero, averagely 60% of the pbd-tagged GFP remained in the well. In a third washing steps this observation was confirmed. While again the main part of the pbd-GFP remained in its original well there could rarely been found any untagged GFP that hadn’t been washed away.
−	In case of the pbd-bound GFP it is striking that after the solution has already been diluted by one washing step, the percentage of protein that can be washed away diminishes. It comes to mind that the massive lost of pbd-tagged GFP after the first washing step might be due to an oversaturation of pbd-GFP in the solution. In this case there would be much more pbd-GFP than place on the plastic surface so that most of the protein could be eluated.	+	In case of the pbd-bound GFP it is striking that after the solution has already been diluted by one washing step, the percentage of protein that can be washed away diminishes.
−	To investigate this phenomenon  we compared different start concentrations of pbd-GFP concerning the amount of pbd-GFP that could be washed away.	+	It comes to mind that the massive lost of pbd-tagged GFP after the first washing step might be due to an oversaturation of pbd-GFP in the solution. In this case [[Image:Freiburg11_picture_2.JPG|300px|left|thumb|Picture 2: % of pbd-tagged GFP that can be eluted in the first washing step. The curve decreases with the amount of the start concentration. If the GFP-pbd solution is not oversaturated a lower amount of this protein can be washed away.]]there would be much more pbd-GFP than place on the plastic surface so that most of the protein could be eluated.To investigate this phenomenon  we compared different start concentrations of pbd-GFP concerning the amount of pbd-GFP that could be washed away.
		+	As shown in picture 2 the percentage of eluted GFP diminishes when the used start concentration is lower. [[Image: pbd_better_than_GFP.jpg|300px|right| thumb | Picture 3: if the start concentration is not oversaturated pbd-coupled is much more resistant to washing steps than GFP alone.]] As the used polystyrene micro titer plates provide only a limited surface for the pbd to bind, the solution shouldn’t be oversaturated with plastic binding protein.In our case the diluted protein could only reach a surface of 91.5 mm<sup>2</sup> per well.
−	[[Image:picture 2 pbd.jpg|300px|thumbPicture 2: % of pbd-tagged GFP that can be eluted in the first washing step. The curve decreases with the amount of the start concentration. If the GFP-pbd solution is not oversaturated a lower amount of this protein can be washed away.]]	+	With a remaining protein amount of ~220 pg/well after three washing steps we estimate that 2,4 pg pbd-tagged protein can be bound per mm<sup>2</sup>.
−		+	In a range of 1-30ng/µL start concentration there remains about seven times more pbd-GFP after washing than "normal" GFP (see picture 3).
−	As shown in picture 2 the percentage of eluted GFP diminishes when the used start concentration is lower. As the used polystyrene micro titer plates provide only a limited surface for the pbd to bind, the solution shouldn’t be oversaturated with plastic binding protein.	+
−	Our scanning of the plastic binding domain did not clearly show that there was more pbd-GFP located in the marginal regions of the wells than in the middle. We assumed that the investigation of the protein concentration via the measurement of the absorbance could give more precise results as the signal doesn’t scatter like fluorescence. We only could do this experiment with pbd-tagged GFP because we didn’t have “normal” really purified GFP.	+
−	For this experiment we mixed 100µL pbd-GFP-protein solution with 200µL Bradford assay. Again we programmed the plate reader to scan 10x10 regions of each well and we performed several washing steps with a new measurement after each step. Looking through the raw data it became clear that there was a higher concentration of pbd-tagged GFP at the well’s margins than in the middle (picture 3).	+
−		+
−		+
−	Picture 3: Detail of a picture of the micro titer plate. The picture is generated by the plate reader and visualizes the allocation of the protein concentration. The color red symbolizes higher protein concentration whereas green color means that there is less protein. From left to right you see the original probe after three washing steps, the first eluate and the  second eluate.	+
	<br/>		<br/>
−	To examine the raw data from the scan we calculated the medians of the most marginal data points and those of the data measured in the center the result is shown in picture 4. As you can see, the protein concentration at the margins is much higher than the concentration in the center. This result indicates that the pbd-GFP binds to the plastic surface of the micro titer plates. Unfortunately we didn’t have any purified GFP without pbd to compare our tagged GFP with. So this experiment doesn’t clearly prove that pbd-GFP binds better to plastic than GFP alone.
−	[[Image:picture 4 pbd.jpg]]
−	Picture 4: Protein concentrations measured via Bradford assay. The red columns show the protein absorbance (~concentration) measured directly at the plastic walls of the micro titer plate’s wells. The green columns show the protein absorbance in the center.
−
		+	====Discussion====
		+	In the experiments described above we could show that in a certain range of start concentration the plastic binding domain (pbd)-coupled GFP was more resistant to elution steps than GFP alone. This result indicates that the pbd-tagged GFP bound stronger to the plastic surface of the microtiter plate than “normal” GFP. The fact that in case of a rather high start concentration the majority of the pbd-GFP was washed away can be explained by the limitation of available binding surface. It seems like the pbd-GFP binds seven times better to the used polystyrene material than GFP alone. The amount of pbd-GFP that can bind to 1 mm<sup>2</sup> is about 2,4 pg. To prove this supposition further measurement and more washing steps should be performed.

---

Latest revision as of 06:21, 26 September 2011

IPTG-inducible Promoter with plastic binding domain-tagged GFP

During phage display experiments small sequences were found that bind to the polystyrene plastic surfaces of the used microtiter plates. The binding was strong enough to resist several washing steps. The sequence binds to plastic due to its high hydropathy.

Usage and Biology

Experimental setup

According to Adey et al. the plastic binding domain (pbd) binds to the polystyrene surface of micro titer plates (96 well plates). To investigate the binding properties of the plastic binding tag we started several spectroscopic assays using a plate reader (FLUOstar Omega) and polystyrene plates (Greiner bio one) To detect the fluorescence of the GFP tagged to the plastic binding domain we used black plates and a well scanning program measuring 10x10 spots in each well of the micro titer plate. We performed several washing steps to find out how much of the proteins can be found in the eluate and how much remains bound on the plate’s surface. To compare the pbd-tagged GFP to a normal GFP without special plastic binding ability we also measured GFP obtained via expression with our diverse [http://2011.igem.org/Team:Freiburg/Results#commons PR] (Promoter-Ribosome-binding-site constructs). To affirm the results obtained by fluorescence spectroscopy used the Bradford assay and screened for different protein concentrations in transparent polystyrene plates. The calibration lines necessary for calculation of protein concentration can be found here.

Results

After the first measurement of the basic fluorescence intensity we transferred the samples onto another well, refilled the exhausted well with PBS and measured both eluate and remaining protein. In a second washing step the liquid was taken out of the first well again and given to another well. This washing was performed a third time, resulting in three eluates and a triply washed well with more or less protein remaining on the walls.

As shown in picture 1, both pbd-tagged and untagged GFP fluorescence decreases after the first washing step. Only 0- 4% of the original concentrations of the untagged GFP and 2-7% of the pbd-tagged GFP remained in the well. After a second washing step there were differences observable between the tagged and the untagged GFP. While the percentage of the remaining “normal” GFP was scattering around zero, averagely 60% of the pbd-tagged GFP remained in the well. In a third washing steps this observation was confirmed. While again the main part of the pbd-GFP remained in its original well there could rarely been found any untagged GFP that hadn’t been washed away. In case of the pbd-bound GFP it is striking that after the solution has already been diluted by one washing step, the percentage of protein that can be washed away diminishes.

It comes to mind that the massive lost of pbd-tagged GFP after the first washing step might be due to an oversaturation of pbd-GFP in the solution. In this case there would be much more pbd-GFP than place on the plastic surface so that most of the protein could be eluated.To investigate this phenomenon we compared different start concentrations of pbd-GFP concerning the amount of pbd-GFP that could be washed away. As shown in picture 2 the percentage of eluted GFP diminishes when the used start concentration is lower. As the used polystyrene micro titer plates provide only a limited surface for the pbd to bind, the solution shouldn’t be oversaturated with plastic binding protein.In our case the diluted protein could only reach a surface of 91.5 mm² per well.

With a remaining protein amount of ~220 pg/well after three washing steps we estimate that 2,4 pg pbd-tagged protein can be bound per mm². In a range of 1-30ng/µL start concentration there remains about seven times more pbd-GFP after washing than "normal" GFP (see picture 3).

Discussion

In the experiments described above we could show that in a certain range of start concentration the plastic binding domain (pbd)-coupled GFP was more resistant to elution steps than GFP alone. This result indicates that the pbd-tagged GFP bound stronger to the plastic surface of the microtiter plate than “normal” GFP. The fact that in case of a rather high start concentration the majority of the pbd-GFP was washed away can be explained by the limitation of available binding surface. It seems like the pbd-GFP binds seven times better to the used polystyrene material than GFP alone. The amount of pbd-GFP that can bind to 1 mm² is about 2,4 pg. To prove this supposition further measurement and more washing steps should be performed.

Sequence and Features