Generator

Part:BBa_K584027

Designed by: Bakul Jitendra Vinchhi, Sarah Veugelen, Katrien Vandermeeren, Yana Hoorne   Group: iGEM11_KULeuven   (2011-09-19)

Constitutive_Promoter + INP(Ice Nucleating Protein Generator)

This part contains the Constitutive Promoter (J23119) with the Ice Nucleating Protein Generator (BBa_K584024) behind it.

The ice nucleating protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. inaZ is the gene responsible for producing the ice nucleation protein.

We would like to thank Prof. Dr. Gregory Gloor , for providing us with a plasmid (pUC1813ICE) which contains the ice nucleating protein gene inaZ.

To know more about the inaZ, it is suggested to read this article describing the usage of pUC1813ICE with the inaZ.


Characterization

Note: To generate supercooled water, ultrapure “milli-Q” water (Millipore Corporation) was filtered over a 0.22µm filter to generate water devoid of nucleating species. All our tests are done in TOP10F' cells

As a negative control, TOP10F’ cells constitutively expressing GFP (brick BBa_K584001) were used.
An overnight preculture was inoculated at an initial OD of 0.1 in 15mL medium, and grown for 5 hours at 37°C. Cells were spun down and resuspended in 1 mL filtered water, after which they were washed twice with 1mL filtered water. The ODs of the resulting cell suspensions were determined and all cells were diluted to the same OD of 10. In the meanwhile, the ethanol bath was set at -6°C, and clean, plastic tubes filled with 5mL filtered water were put in the chilled bath to create supercooled water at -6°C. For the INP test, the same amount of INP-expressing or control cells were added to the 5mL supercooled water and checked for ice nucleating activity (see videos).

As can be seen on the videos, addition of cells expressing INP clearly induced ice crystallization, while adding the same amount of control cells to supercooled water does not. Hence, our INP construct works! :-)





The UGent Belgium 2016 iGEM team improved this part by removing 2 BsaI restriction sites and substituting the J23119 promoter for a weaker constitutive promoter in order to decrease toxicity and improve genetic stability, creating BBa_K1896011.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 707
    Illegal NgoMIV site found at 2219
    Illegal NgoMIV site found at 2363
    Illegal NgoMIV site found at 2627
    Illegal NgoMIV site found at 2771
    Illegal NgoMIV site found at 2819
    Illegal NgoMIV site found at 2939
    Illegal AgeI site found at 478
    Illegal AgeI site found at 1883
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 653
    Illegal BsaI.rc site found at 3399
    Illegal SapI.rc site found at 299


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