Difference between revisions of "Part:BBa K1055001:Experience"

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===Applications of BBa_K1055001===
 
===Applications of BBa_K1055001===
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When tried to fuse to the Tar receptor of ''E. coli''  C-terminally we encountered major clonation problems since our methods were not able to acces the DNA. After numerous trials and several methods, we performed an "m-fold" analysis of our hypothetical DNA construct and obtained information about a hypothetical secondary DNA structure. It is thermodynamically stable ( We derived dG =  - 318 kj/mol). Please take a look below:
  
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LSSmOrange is an orange fluorescent protein that we want to use for FRET, we us it as a donor. LSSmOrange has an excitation maximum by 437 nm and an emission maximum by 572 nm.  The excitation maximum is at 415 nm, another at 453 nm and our measured emission maximum is at 564 nm. This aberration changes nothing on the FRET system. The excitation maximum is big enough to stimulate [https://parts.igem.org/Part:BBa_K1055000 mKate], the other fluorescent protein. Of course, we checked also the emission and excitation of our BL21(DE3) cells (data not shown) and we can eliminate the theories that these cells disturb this florescence measurement.
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[[Image:mfold_Lssmorange.png|center|400px|thumb|Hypothetical DNA secondary structure of Tar-LssmOrange fusion protein which might be the reason for continously failing clonation attempts. We derived dG = - 318 kj/mol]]
  
 
===User Reviews===
 
===User Reviews===
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[[Image:LSSmOrange_Diagramm.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
 
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[[Image:Grow_log.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
 
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[[Image:LSSmOrange_bac.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
 
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[[Image:mKate-LSSmOrange_FRET_Pair.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
 

Latest revision as of 18:09, 12 October 2013

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Applications of BBa_K1055001

When tried to fuse to the Tar receptor of E. coli C-terminally we encountered major clonation problems since our methods were not able to acces the DNA. After numerous trials and several methods, we performed an "m-fold" analysis of our hypothetical DNA construct and obtained information about a hypothetical secondary DNA structure. It is thermodynamically stable ( We derived dG = - 318 kj/mol). Please take a look below:

Hypothetical DNA secondary structure of Tar-LssmOrange fusion protein which might be the reason for continously failing clonation attempts. We derived dG = - 318 kj/mol

User Reviews

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