Composite

Part:BBa_K215107

Designed by: Josef Dunbar   Group: iGEM09_Washington   (2009-09-30)
Revision as of 02:50, 19 October 2009 by Siegeljb (Talk | contribs) (→‎Usage and Biology)

Extracellular Secretion System

This part is made of 3 genes: prtD, prtE, and prtF, that constitute a type I Erwinia chrysanthemi secretion system. The operon is expressed from a strong constitutive promoter, BBa_J23100, and has the translational terminator BBa_B0014. In PSB3T5.

Used to secrete proteins containing prtB C-terminal tag. The prtB C-terminal tag is built into the protein generator [BBa_K215002]. Any protein of interest can be inserted into the protein generator and then secreted when used in conjunction with this secretion system.


Usage and Biology

For full characterization data on this part, please see the [http://2009.igem.org/Team:Washington/Project/Secretion University of Washington 2009 iGEM project page].


To test this secretion system's functionality, it was transformed into cells (strain BL21 lacIq) also containing a plasmid with part [BBa_K215010] (IPTG-induced GFP fusion protein containing prtB secretion tag at C-terminus). The cells were then grown in a large culture and expression induced via IPTG. After a period of growth, the cells were then spun down, and protein was purified from the supernatant using [http://2009.igem.org/Team:Washington/Notebook/IMAC_protocol IMAC purification] to separate the tagged GFP. The fluorescence in the purified supernatant was then used to quantify the amount of target GFP released from the cells into the media based on a standard curve of GFP that was previously generated from BBa_K215002.

  • Tagged GFP in Media The red data set corresponds to cells containing this secretion system plasmid and part BBa_K215011 (fusion OpdA with prtB tag), the blue data set corresponds to cells containing a promoter-less version of the secretion system and part BBa_K215010 (fusion GFP with prtB tag), and the green data set corresponds to cells containing this secretion system and part BBa_K215010. As the plot shows, a substantial amount of the tagged GFP was released into the media even when the secretion construct did not have a promoter. This result suggests that the tagged GFP in the media was not the result of the secretion, but is rather an artifact.

Based on these results, it has yet to be shown that the Secretion system is functioning. However, this system has been shown to work properly in the literature, and there are many parameters that we have yet to reproduce and optimize. Proteins that have been secreted by this Type I system and other similar ones include GFP, lipase, Trichoderma harzianum endochitinase, trout growth hormone, ompC, and lacZ. This causes us to believe that this part, which has been sequenced verified, should work given a little more tweaking, to see our ideas see the UW iGEM 2009 [http://2009.igem.org/Team:Washington/Future Furture Directions].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1487
    Illegal BsaI.rc site found at 1133
    Illegal BsaI.rc site found at 3737


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