Difference between revisions of "Part:BBa K3815001"
Line 21: Line 21:
 
</ul>
 
</ul>
 
<h3><font size="4.5">Purification </font></h3>
 
<h3><font size="4.5">Purification </font></h3>
−
1.<i>E.coli</i> which expressed this part were lysed with sonification.<br>
+
1.<i>E.coli</i> which expressed this part were lysed with sonification.
 
2.When this fused protein are produced,it self-assembles and precipitates.
 
2.When this fused protein are produced,it self-assembles and precipitates.
 
3.This aggregate is collected by centrifugation.
 
3.This aggregate is collected by centrifugation.
 
4.Adding DTT to this aggregate, the targeted protein peptides is cut out by the cleavage of intein for one whole day.  
 
4.Adding DTT to this aggregate, the targeted protein peptides is cut out by the cleavage of intein for one whole day.  
 +
<br>
 +
<br>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 15:41, 19 October 2021


CecropinA-Mxe GryA intein-PT-linker-ELK16

Usage and Biology

We used this part for purfication of antimicrobial peptide, CecropinA. This is derived from a moth. This is the antimicrobial peptide that can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water. This part is used for ELK16 system to purify the peptides. When this fused protein is produced, the part of ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, we cut the N terminal of Mxe GyrA intein. Then, we can get the targeted protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 814
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 67
    Illegal AgeI site found at 346
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification

SDS-PAGE of purified peptide

Expression

  • Cells were grown in 1000ml LB media at 37oC.
  • when the OD exceeded 0.35, IPTG was added to induce the peptide expression.

Purification

1.E.coli which expressed this part were lysed with sonification. 2.When this fused protein are produced,it self-assembles and precipitates. 3.This aggregate is collected by centrifugation. 4.Adding DTT to this aggregate, the targeted protein peptides is cut out by the cleavage of intein for one whole day.