Difference between revisions of "Part:BBa K4015007"
Line 9: Line 9:
  
 
<p>We created p47-laccase-Lcp, p47-laccase-Lcp-HlyA, p47-laccase-HlyA, and pET28-Lcp-HlyA as the control to test our hypothesis. In the results, the control group (Figure 4A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Figure 4B) secretion happened which implies that laccase by itself can act as a secretion agent; for p47-laccase-HlyA, no secretion happened, and we suspect that HlyA and laccase might suppress each other’s activity; however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved, showed by Figure 4. As shown by figure 4, CotA-Lcp-HlyA has achieved successful secretion.  
 
<p>We created p47-laccase-Lcp, p47-laccase-Lcp-HlyA, p47-laccase-HlyA, and pET28-Lcp-HlyA as the control to test our hypothesis. In the results, the control group (Figure 4A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Figure 4B) secretion happened which implies that laccase by itself can act as a secretion agent; for p47-laccase-HlyA, no secretion happened, and we suspect that HlyA and laccase might suppress each other’s activity; however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved, showed by Figure 4. As shown by figure 4, CotA-Lcp-HlyA has achieved successful secretion.  
 +
 +
 
https://static.igem.org/mediawiki/parts/4/45/T--KEYSTONE--p23.png
 
https://static.igem.org/mediawiki/parts/4/45/T--KEYSTONE--p23.png
 +
  
 
Fig  4.    A. Strain containing p28a-lcp-hlyA; no coloration observed. B. Srtain containing p47-laccase-lcp; coloration was observed, indicating success in secretion. C. Strain containing p47-laccase-hlyA; no coloration observed. D. Strain containing p47-laccase-lcp-hlyA; coloration was observed, indicating success in secretion. 
 
Fig  4.    A. Strain containing p28a-lcp-hlyA; no coloration observed. B. Srtain containing p47-laccase-lcp; coloration was observed, indicating success in secretion. C. Strain containing p47-laccase-hlyA; no coloration observed. D. Strain containing p47-laccase-lcp-hlyA; coloration was observed, indicating success in secretion. 

Revision as of 11:01, 20 October 2021


CotA-Lcp-HlyA

CotA-Lcp-HlyA is composed of three basic parts, Lcp1VH2, HlyA and CotA. HlyA and Lcp are connected by Gibson assembly in order to allow Lcp to be secreted outside the cell through HlyA,and assembling CotA on the composite part can help to indicate the secretion.


Usage and Biology

We created p47-laccase-Lcp, p47-laccase-Lcp-HlyA, p47-laccase-HlyA, and pET28-Lcp-HlyA as the control to test our hypothesis. In the results, the control group (Figure 4A), as expected, showed no sign of coloration due to the absence of laccase; in the sample containing p47-laccase-Lcp (Figure 4B) secretion happened which implies that laccase by itself can act as a secretion agent; for p47-laccase-HlyA, no secretion happened, and we suspect that HlyA and laccase might suppress each other’s activity; however, when laccase and HlyA are positioned at the two ends of Lcp, this conflict is resolved, showed by Figure 4. As shown by figure 4, CotA-Lcp-HlyA has achieved successful secretion. T--KEYSTONE--p23.png Fig  4.    A. Strain containing p28a-lcp-hlyA; no coloration observed. B. Srtain containing p47-laccase-lcp; coloration was observed, indicating success in secretion. C. Strain containing p47-laccase-hlyA; no coloration observed. D. Strain containing p47-laccase-lcp-hlyA; coloration was observed, indicating success in secretion. 


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1348
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 286