Team:Wisconsin-Madison/parts 2


T5 Exonuclease

T5 exonuclease originates from the D15 gene of bacteriophage T5, which commonly infects bacteria such as Escherichia coli. The enzyme has been proven to increase the DNAse activity of E. coli, selectively degrading linear DNA in a 5’ to 3’ fashion. T5 exonuclease has been shown to have no effect on double stranded circular DNA (Sayers 1991). T5 exonuclease exhibits optimal function at temperatures near 37°C, and thus is deactivated during the Gibson Assembly (Gibson, 2009). This inactivation is necessary to prevent complete hydrolyzation of the double-stranded DNA.

Like the Taq Ligase gene, the T5 exonuclease gene is also within a kanamycin resistant PET-ALM1 plasmid, downstream of the T7 promoter, upstream of the T7 terminator. Thus, it can be used with an inducible T7 expression system to express the enzyme. The protein has an N-Terminus hexa-histidine tag, joined to the wild type structure by a thrombin cleavage motif.

Activity of T5 exonuclease. Lane1-single stranded DNA. Lane 2-RFIII, RFIV, SS DNA. Lane 3-reaction with 600 ng T5 exonuclease. Lane 4-reaction with 60 ng T5 exonuclease. Lane 5-reaction with 6 ng T5 exonuclease. All reactions occurred at 37°C for 30 minutes. (Sayers 1991)