E.Coli derived camelid antibodies as a sensor for p53 in saliva

Introducing competence into E. coli using phi 29 DNA packaging motor

We became interested in this topic because E. coli is one of the most popular model organisms used for laboratory work. However, E. coli cells possess a weak tendency to uptake DNA from the surroundings, i.e. it shows a weak competence. As you know, competence can be induced by two common methods: electroporation and chemical transformation using divalent cations. Electroporation requires extra equipment as electroporator, while chemical transformation takes at least one day preparation. Therefore, we suggest to incorporate hexameric ATPasegp 16 from DNA packaging motor of phi29 virus into inner and outer membrane of E. coli using signal leading peptides from the iGEM distribution kit.

DNA packaging motor. This protein can be found in virus phi29 from B. subtilus. The protein belongs to superfamily of AAA+ motors (ATP-ases associated with diverse cellular activities). Gp16 functions as ATPase during DNA packaging. It has been observed that gp16 binds DNA without sequence specificity, but specifically to pRNA,which implies that the binding properties of gp16 are important determining factors for the biological activity of gp16.

The advantage of phi29 DNA packaging motor is that has a larger channel diameter, and hence it can easily translocate both dsDNA and ssDNA and large cavity size of this channel enables relatively easy insertion of functional moieties with desirable characteristics via conjugation to amino acid residues lining channel cavity or at terminal ends for diverse applications. However, the disadvantage of this protein for our idea is that it can transport only linear DNA.

Our aim:To express gp16 of DNA packaging motor from phi29 on the surface of E. coli and to determine the uptake of DNA plasmids by the modified bacterium.

References

Schwartz C, De Donatis GM, Fang H, Guo P. (2013). The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily. Virology. 443: 20–27.