Difference between revisions of "Part:BBa K1055000:Experience"

Latest revision as of 18:20, 12 October 2013

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Applications of BBa_K1055001

When tried to fuse to the CheB signaling protein 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 = - 232 kj/mol). Please take a look below:

Hypothetical DNA secondary structure of CheB-mKate fusion protein which might be the reason for continously failing clonation attempts. We derived dG = - 232 kj/mol

User Reviews

UNIQ10b5743ce403db90-partinfo-00000000-QINU UNIQ10b5743ce403db90-partinfo-00000001-QINU

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 how you used this part and how it worked out.
-===Applications of BBa_K1055000===
+===Applications of BBa_K1055001===
+When tried to fuse to the CheB signaling protein 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 =  - 232 kj/mol). Please take a look below:
+[[Image:CMK_scstr.png|center|400px|thumb|Hypothetical DNA secondary structure of CheB-mKate fusion protein which might be the reason for continously failing clonation attempts. We derived dG =  - 232 kj/mol]]
 ===User Reviews===
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 <!-- DON'T DELETE --><partinfo>BBa_K1055000 EndReviews</partinfo>
-'''Characterization mKate'''
-mKate2 is a red fluorescent protein that we want to use as a acceptor for FRET. According to Evrogen [2] mKate2 has an excitation maximum at 588 nm and an emission maximum at 633 nm. The emission maximum is big enough to get stimulated by LSSmOrange, 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.
-[[Image:Spectra_mKate.png|400px|thumb|Figure 1. '''Excitation Spectrum (dashed line) and emission spectrum (solid line) of mKate with marked maximums''' Induced at time=0h.]]
-[[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.]]
-[[Image:mKate_bac.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]
-[[Image:Grow_log.png|400px|thumb|Figure 1. '''Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression.''' Induced at time=0h.]]