Team:KULeuven/Project Results

From 2009.igem.org

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(Project Results)
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Our Results can be roughly divided in two sections, the biobrick assembly from starting parts in order to create our (test)-devices and secondly the characterizations of a part.
Our Results can be roughly divided in two sections, the biobrick assembly from starting parts in order to create our (test)-devices and secondly the characterizations of a part.
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First and foremost we succesfully assembled the following biobricks and sent them to the registry:
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First and foremost the following biobricks were succesfully assembled and to the registry:
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Next, the effect of blue light on this promoter was examined.
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Next, the effect of blue light on this promoter was examined. As shown on the graph below, blue light irradiation has a significant effect on the strength of the promoter. However, aside from light, temperature also has an important effect on promotor strenght (possibly even bigger then light). For instance, when using low copy plasmids, temperature will influence the promoter/repressor ratio. Cells grown at 37°C will have a too large effect on the promoter, resulting in constitutive activity. At 16°C, there will be more repressor and more receptor, however the low temperature will put the receptor in its active state causing it to inhibit the repressor from doing its job. When grown at 25°C the ratio between repressor and receptor is better, without light irradiance the receptor/represssor are in there ground state resulting in a good repression of the promoter.  
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As this graph shows, blue light does have a significant effect on the promoter. However, aside from light, temperature is a very important influence (if not a bigger one). For instance, when working in low copy plasmids, temperature will influence the promoter/repressor ratio. Cells grown at 37°C will have too much effect on the promoter, resulting in constitutive activity. At 16°C, there will be more repressor and receptor influence, but the low temperature will put the receptor in its active state thus inhibiting the repressor from doing its job. When grown at 25°C the ratio is better and the receptor/represssor are still in there ground state leading to a good repression of the promoter.  
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Our experimental set-up:  
Our experimental set-up:  
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Liquid cultures were ented (and re-ented in the evening) and grown on two different temperatures: 37° and 25°C.  
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Liquid cultures were ented (and re-ented in the evening) and grown on two different temperatures: 37° and 25°C.  Note: enting of the cultures needs to be done in a dark room (red light)and it might be best to use medium that was put at 4°C overnight.
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From each of these growth conditions some of the cultures were put in 16° and the other at 25°C. Each of these groups were then again divided in a group that was lit with blue light and a group that wasn't.  
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From each of these growth conditions some of the cultures were put in 16° and the other at 25°C. Each of these groups were then again divided in a group that was exposed to blue light and a group that was not exposed to light.  
Measurements were done at following hours: 0h;0,5h;1,5h;3h;4,5h;6h;8h.  
Measurements were done at following hours: 0h;0,5h;1,5h;3h;4,5h;6h;8h.  
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This was done for our ligation of blp with GFP in three different vectors (pSB3K3, pBR322 and pSB1A2).  
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The construct used for our test was a ligation of the blue light promotor in front of GFP using three different vectors (pSB3K3, pBR322 and pSB1A2). (An example of a sample name is: 37/16/NB/LigA, grown at 37°, put in 16°, not lit and in vector pSB1A2. )
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An example of a sample name is: 37/16/NB/LigA, grown at 37°, put in 16°, not lit and in vector pSB1A2.  
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Note: enting of the cultures needs to be done in a dark room (red light)and it might be best to use medium that was put at 4°C overnight.
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This experiment was repeated some times and variations were done:
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only growing cultures on 25°C and lighting them at room temperature
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{{Team:KULeuven/Common2/PageFooter}}

Revision as of 12:17, 20 October 2009


Project Results

Our Results can be roughly divided in two sections, the biobrick assembly from starting parts in order to create our (test)-devices and secondly the characterizations of a part.

First and foremost the following biobricks were succesfully assembled and to the registry:

Partnumber Description
Antisense key
Sam5 + rbs
virA
VirG
VirB
Blue light promoter
Ech+fcs
Blp + GFP
Blp + RFP
Ech + Fcs + terminator
Promotor+ Ech + FCS + terminator
Sam8 +Sam8
Sam8 + Sam5 + promoter
Sam8+ Sam5 + promoter + comt
Promotor + Sam8 + Sam5 + comt + terminator
lock
Sam8 + RBS
Ech + rbs
Fcs + rbs


Furthermore, the blue light promotor was extensively characterized. The strength of the promoter was compared to the standard iGEM promoter, , using the protocol from the iGEM measurement kit.

Comparison of the blue light promoter (blue) to a standard promoter strength (orange) in RPU. The blue light promoter shows at least half the strength of the standard promoter, meaning it can be classified as a weak promoter

Next, the effect of blue light on this promoter was examined. As shown on the graph below, blue light irradiation has a significant effect on the strength of the promoter. However, aside from light, temperature also has an important effect on promotor strenght (possibly even bigger then light). For instance, when using low copy plasmids, temperature will influence the promoter/repressor ratio. Cells grown at 37°C will have a too large effect on the promoter, resulting in constitutive activity. At 16°C, there will be more repressor and more receptor, however the low temperature will put the receptor in its active state causing it to inhibit the repressor from doing its job. When grown at 25°C the ratio between repressor and receptor is better, without light irradiance the receptor/represssor are in there ground state resulting in a good repression of the promoter.

Effect of blue light irradiance on promoter strength. Cells grown under blue light irradiance (blue) show a stronger expression compared to cells grown in the dark (grey)

Our experimental set-up:

Liquid cultures were ented (and re-ented in the evening) and grown on two different temperatures: 37° and 25°C. Note: enting of the cultures needs to be done in a dark room (red light)and it might be best to use medium that was put at 4°C overnight. From each of these growth conditions some of the cultures were put in 16° and the other at 25°C. Each of these groups were then again divided in a group that was exposed to blue light and a group that was not exposed to light. Measurements were done at following hours: 0h;0,5h;1,5h;3h;4,5h;6h;8h. The construct used for our test was a ligation of the blue light promotor in front of GFP using three different vectors (pSB3K3, pBR322 and pSB1A2). (An example of a sample name is: 37/16/NB/LigA, grown at 37°, put in 16°, not lit and in vector pSB1A2. )