Coding

Part:BBa_K3894012

Designed by: Jiaqi Zhang   Group: iGEM21_NEFU_China   (2021-10-17)


Klenow.mut-SH3
The iGEM 2021 NEFU_China created the D424A mutant of Klenow (BBa_K3894022) and constructed Klenow.mut-SH3 that theoretically lost its 3'→5' exonuclease activity.

Description

The iGEM 2021 NEFU_China created the D424A mutant of Klenow (Klenow-mut) and constructed Klenow.mut-SH3 that theoretically lost its 3'→5' exonuclease activity[1]. This mutant could ensure the complete single strand replacement without possible degradation of the displaced DNA. Klenow still keeps the 5'→3' polymerase activity and 3'→5' exonuclease activity of DNA polymerase I, but lacks the 5'→3' exonuclease activity of the complete enzyme. We added Src homology Domain 3 (SH3) sequence to the N-terminus of Klenow, which can bind to proline-rich ligand and allow multiprotein assembly in vitro. Based on this strategy, our team proposed an in vitro protein assembly approach, which may help future teams who encounter difficulties in expressing and purifying large proteins using bacterial systems. Importantly, the relatively small sizes of SH3 and slig compared to the components of the Spy system may better meet the requirements in specific scenarios.

Experience

Klenow sequence was synthesized and successfully expressed and purified in our laboratory. Klenow have strong chain replacement activity, and can replace the single chain containing the nick to obtain the single strand primer we need. Meanwhile, EMSA was used to compare the exonuclease activity of Klenow and Klenow.mut.

Figure 1. SDS-PAGE analysis of recombinant Klenow.mut-SH3. The molecular weight of the Klenow.mut-SH3 protein is 112.6 kDa.| 1 and 2. Eluent of His×6-tagged Klenow.mut from Ni-NTA agarose beads.

Figure 2. DNA agarose gel of the FAM-labeled probe after incubated with Klenow, Klenow.mut and Phi29. | 1. Klenow + FAM-probe. 2. Klenow.mut + FAM-probe. 3. Phi29 + FAM-probe. 4. FAM-probe alone. Xxx µg of each enzyme and xxx pmol of the probe were used. Control: probe only.

The iGEM 2021 NEFU_China used EMSA to compare the exonuclease activity of Klenow, Klenow.mut and Phi29. Compared to the control sample, both Klenow and Klenow.mut could generate fast migrated bands, but Klenow.mut bands were more diffused than these of Klenow, suggesting that the mutation could attenuate the exonuclease activity of Klenow. Based on these results, we concluded that the Klenow.mut would be the best choice among the three enzymes in mediating the ssDNA replacement.

Reference

[1]Pinky, Kukreti, Kamalendra, et al. Identification of a new motif required for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I (Klenow fragment): the RRRY motif is necessary for the binding of single-stranded DNA substrate and the template strand of the mismatched duplex.[J]. Journal of Biological Chemistry, 2008.  



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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