Difference between revisions of "Part:BBa K331030:Experience"

(→‎User Reviews)
(→‎Method)
Line 25: Line 25:
 
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.
 
This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (<i>Escherichia coli</i> DH5&alpha;), N-terminal tagged, and C-terminal tagged YFP were compared.
 
<br><br>
 
<br><br>
 +
 +
<html>
 +
<center>
 +
<body>
 +
<table border="0" cellpadding="8"  width="28%"  style="background-color:#000000">
 +
 +
<tr>
 +
<th>
 +
<img src="" width="350"/>
 +
</th>
 +
 +
<tr><th>
 +
<img src="" width="350"/>
 +
</th>
 +
</tr>
 +
 +
</table>
 +
</body>
 +
</center>
 +
</html>
  
 
===Results===
 
===Results===

Revision as of 20:19, 27 October 2010

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_K331030

In order to further characterize the C-terminal and N-terminal oligoarginine tag (BioBricks BBa_K249005 and BBa_K249004 respectively) and investigate the effect their placement on protein stability, yellow fluorescent proteins (YFP) with the oligoarginine fused to either the C-terminus (BBa_K331023) or N-terminus (BBa_K331022) (and preceded by a ribosomal binding site – BBa_B0034) were synthesized. We used our Red/White 3-Antibiotic assembly method to add a tetracycline repressible promoter (BBa_R0040) for constitutive expression of the fusion protein. This addition generated BioBricks BBa_K331031 and BBa_K331030 for the C-terminal tagged and N-terminal tagged YFP respectively.

User Reviews

UNIQb594ef507705a2f1-partinfo-00000009-QINU UNIQb594ef507705a2f1-partinfo-0000000A-QINU

Method

The BioBrick containing plasmid was transformed into Escherichia coli DH5α cells. These cells were grown to an OD600 of approximately 0.7, and diluted 1:10 with MilliQ H2O immediately prior to analysis by fluorescent spectroscopy.

This dilution of cells was excited at 517 nm, and the emission spectra was read from 522 nm to 650 nm. Fluorescence at 524 nm (emission maxima of YFP) of control cells (Escherichia coli DH5α), N-terminal tagged, and C-terminal tagged YFP were compared.

Results

N-terminal tagged YFP did not have substantially more fluorescence than control cells. Cells expressing C-terminal tagged YFP had ten times more fluorescence than control cells and cells expressing N-terminal tagged YFP. 900px

Conclusion

Our results are consistent with the data reported by Bachmair et al. in that the placement of arginine residues at the N-terminus of our YFP results in no observable fluorescence over control cells. Assuming that transcription of this K331030 and K331031 are equivalent, these data suggest that the N-terminal oligoarginine is reducing the half-life of the protein to which it is fused, ie YFP.

Reference

1Bachmair A., Finley D., Varshavsky A. (1986), In Vivo Half-Life of a Protein Is a Function of Its Amino-Terminal Residue. Science 234. 4773 179-186.