Difference between revisions of "Part:BBa I6211:Experience"

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===iGEM Stockholm 2017===
 
===iGEM Stockholm 2017===
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Our team wanted to test how the osmotic pressure promoter (BBa_R0083) worked by measuring the YFP fluorescence at different OD600 values and different concentrations of sucrose and NaCl.
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We first cloned the biobrick (BBa_I6211) into a low copy number plasmid (pSB4A5) to yield higher levels of fluorescence. Then we started our osmotic test by cultivating TOP10 cells, transformed with the successful clonings, in sucrose and salt gradients and thereafter measured fluorescence of YFP at each OD600 from 0.1 to 0.6, expecting to see higher levels of fluorescence in higher sucrose and salt concentrations. Sucrose concentrations of 5,10 and 15% and NaCl concentrations of 0.00625, 0.0125, 0.0025, 0.05 and 0.1% were used. We used both negative and positive controls in the highest concentrations of sucrose and NaCl and in a 0% sample. Our negative control was untransformed TOP10 cells and positive control TOP10 cells transformed with constitutively expressed YFP (BBa_K592101).
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However, the obtained results from the fluorescence measurements were vague. First of all, the negative controls showed higher fluorescence than some of the samples. This is further demonstrated in figure 1. As the negative control contained no YFP, we concluded that the bacteria itself have a lot of background fluorescence. Second, we observed no trend regarding increasing fluorescence with rising osmotic pressure, neither in sucrose nor in the salt gradient. We could therefore not draw any conclusions regarding the activity of the OmpR promoter when using YFP as reporter and does not recommend this biobrick to be used for this purpose. We continued our test with a similar biobrick, BBa_M30011, which uses RFP instead and we got much better results. [[File:YFP test.png|600px|thumb|left|Figure 1]]
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<I>iGEM Stockholm 2017</I>
 
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Our team wanted to test how the osmotic pressure promoter (BBa_R0083) worked by measuring the YFP fluorescence at different OD600 values
 
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and different concentrations of sucrose and NaCl.
 
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We first cloned the biobrick (BBa_I6211) into a low copy number plasmid (pSB4A5) to yield higher levels of fluorescence. Then we started our osmotic test by cultivating TOP10 cells, transformed with the successful clonings, in sucrose and salt gradients and thereafter measured fluorescence of YFP at each OD600 from 0.1 to 0.6, expecting to see higher levels of fluorescence in higher sucrose and salt concentrations. We used both negative and positive controls in the highest concentrations of sucrose and NaCl and in a 0% sample. Our negative control was untransformed TOP10 cells and positive control TOP10 cells transformed with constitutively expressed YFP (BBa_K592101).
 
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However, the obtained results from the fluorescence measurements were vague. First of all, the negative controls showed higher fluorescence than some of the samples. This is further demonstrated in figure XX. As the negative control contained no YFP, we concluded that the bacteria itself have a lot of background fluorescence. Second, we observed no trend regarding increasing fluorescence with rising osmotic pressure, neither in sucrose nor in the salt gradient. We could therefore not draw any conclusions regarding the activity of the OmpR promoter when using YFP as reporter and does not recommend this biobrick to be used for this purpose. We continued our test with a similar biobrick, BBa_M30011, which uses RFP instead and we got much better  results.
 
 
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Latest revision as of 11:49, 18 October 2017


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I6211

iGEM Stockholm 2017

Our team wanted to test how the osmotic pressure promoter (BBa_R0083) worked by measuring the YFP fluorescence at different OD600 values and different concentrations of sucrose and NaCl.

We first cloned the biobrick (BBa_I6211) into a low copy number plasmid (pSB4A5) to yield higher levels of fluorescence. Then we started our osmotic test by cultivating TOP10 cells, transformed with the successful clonings, in sucrose and salt gradients and thereafter measured fluorescence of YFP at each OD600 from 0.1 to 0.6, expecting to see higher levels of fluorescence in higher sucrose and salt concentrations. Sucrose concentrations of 5,10 and 15% and NaCl concentrations of 0.00625, 0.0125, 0.0025, 0.05 and 0.1% were used. We used both negative and positive controls in the highest concentrations of sucrose and NaCl and in a 0% sample. Our negative control was untransformed TOP10 cells and positive control TOP10 cells transformed with constitutively expressed YFP (BBa_K592101).

However, the obtained results from the fluorescence measurements were vague. First of all, the negative controls showed higher fluorescence than some of the samples. This is further demonstrated in figure 1. As the negative control contained no YFP, we concluded that the bacteria itself have a lot of background fluorescence. Second, we observed no trend regarding increasing fluorescence with rising osmotic pressure, neither in sucrose nor in the salt gradient. We could therefore not draw any conclusions regarding the activity of the OmpR promoter when using YFP as reporter and does not recommend this biobrick to be used for this purpose. We continued our test with a similar biobrick, BBa_M30011, which uses RFP instead and we got much better results.
Figure 1


UNIQeca35708cd654f59-partinfo-00000000-QINU

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

UNIQeca35708cd654f59-partinfo-00000002-QINU