Generator

Part:BBa_K117001

Designed by: Nguyen Xuan Hung   Group: iGEM08_NTU-Singapore   (2008-10-07)

ColicinE7 with Immunity

This gene sequence once expressed will produce colicin E7. This is an endonuclease with random cutting site and can kill various strains of EHEC bacteria. However, inside the host cell, immediately after being produced, its toxicity is inhibited by Immunity protein (which is also expressed at the same time). These 2 proteins form a complex which is harmless to the host cell. Under SOS response, the host cell produces lysis protein, which cause the cell to lyze. At the same time, lysis protein remove Immunity protein from the complex with colE7. As a result, colE7 regains its toxicity and ability to kill the EHEC bacteria nearby.
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Part Description

2.1 What is it needed for? Function.

Colicin E7 (ceaE7) belongs to the E-group colicins (El to E9) which are bacterial toxins (bacteriocins) that bind to the vitamin B12 receptor and translocate across the sensitive bacteria’ membranes to induce cell deaths. They are further classified into 3 types, based on the mechanism of attacking target bacteria: RNAase-type colicins (such as E3 and E6); pore-forming colicin (E1); and DNAase-type colicins (E2, E7, and E8). Among the strains of bacteria that are sensitive to ColE7 is E. coli O157:H7 which causes severe food-borne diseases. Previous reports showed that ColE7 was able to attack all 22 O157:H7 strains tested (Schamberger, 2002). As our project targets on the EHEC strains, ColE7 stands out as the best bacteriocin.

2.2 How does it work?

As a DNAase-type colicin, ColE7 nonspecifically breaks the DNA of sensitive cells. It is capable of cutting both single strands and double strands of DNA. To protect the host cell from bactericidal activity of ColE7, the expression of E7 is immediately followed by production of the immunity protein (ceiE7) which binds specifically to the DNAase domain of ColE7 and thus neutralizes its toxicity toward the host cell.

2.3 How do you use it?

Expression of ColE7 in the host cell is naturally regulated by "SOS" response. The response produces ColE7-ImmE7 complex, followed by lysis protein. This protein removes immunity protein from ColE7 and also causes the host cell to lyse. As a result, ColE7 is released into the external cell medium and able to attack sensitive bacteria. For more information on the lysis gene (celE7), please visit part BBa_K117000. The aim of our project is to be able to control the production of ColE7-ImmE7 and the cell lysis separately. Hence, our bacteria are engineered to carry 2 separate functions: production of ColE7 together with ImmE7 (to protect themselves) and production of lysis protein to cause cell lysis. The ColE7-ImmE7 gene is the main part of our production system. Bacteria with this system plasmid are able to produce ColE7-ImmE7 when induced by lactose.

References:

  • Schamberger, G.,and F.Diez-Gonzalez.,Selection of recently isolated colicinogenic Escherichia coli strains inhibitory against E coli O157:H7,Food Prot.,2002,1381-1387.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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