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

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how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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===Applications of BBa_K1055000===
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===Applications of BBa_K1055001===
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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:
  
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''Characterization mKate''
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[[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]]
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[[Image:MKate-LSSmOrange_FRET_Pair.png|400px|thumb|Figure 2. '''Overlay of excitation spectrum (dashed line) and emission spectrum (solid line) of mKate and LSSmOrange ''']]
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mKate2 is a red fluorescent protein that we want to use as a acceptor for FRET. According to [http://www.evrogen.com/products/basicFPs.shtml Evrogen] 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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1055001 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.
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[[Image:Spectra_mKate.png|400px|thumb|Figure 1. '''Excitation Spectrum (dashed line) and emission spectrum (solid line) of mKate with marked maximums''' ]]
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[[Image:mKate_bac.png|400px|thumb|Figure 3. '''Bl21-DE3 cells with mKate ''']]
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[[Image:Grow_log.png|400px|thumb|Figure 4. '''Semi-logarithmic growth curves of BL21DE3 WT, BL21DE3 mKate and BL21DE3 LSSmOrange. The ln (OD600 values) are plotted against the time [min].''']]
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===User Reviews===
 
===User Reviews===

Latest revision as of 18:20, 12 October 2013

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_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

UNIQ3557fa8ee420133f-partinfo-00000000-QINU UNIQ3557fa8ee420133f-partinfo-00000001-QINU