Coding

Part:BBa_K3894022

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


Klenow-SH3
Klenow is mainly used for single strand replacement in our project.

Description

Klenow is mainly used for single strand replacement in our project. DNA polymerase I is from E. coli, and the complete enzyme contains two fragments with molecular weights of 76 and 34 kDa. The large fragment, named as Klenow, retains the 5’-to-3’ DNA synthesis activity and 3’-to-5’ exonuclease activity, but lacks the 5’-to-3’ exonuclease activity of the complete enzyme[1]. We added the Src homology domain 3 (SH3) sequence to the C-terminus of the Klenow, which can bind to proline-rich ligands for multiprotein assembly in vitro. Based on this design, we proposed an in vitro protein assembly method, which could provide ideas and useful parts for future iGEM teams with difficulties in purifying macromolecular proteins. In addition, the SH3 and slig sequences have smaller molecular weights than the components of the Spy system, which can better meet the design requirements in certain scenarios.

Experience

The coding sequence of the Klenow was synthesized, robustly expressed, and successfully purified in our studies. Meanwhile, we used electrophoretic mobility shift assay (EMSA) to compare the performance of Klenow, Klenow.mut (BBa_K3894012 ) and Phi29（BBa_K3894014） in generating nicks on double-stranded DNA.

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

Figure 2. EMSA to verify the ssDNA replacing activity of the Klenow. | 1. With both Nb.BbvCI and Klenow. 2. Without Nb.BbvCI. 3. Without Klenow. 4. FAM-probe alone. 5. Annealed FAM-probe/c-probe.

The iGEM 2021 NEFU_China tested the ssDNA replacing activity of Klenow by EMSA. In the presence of both Nb.BbvCI and Klenow, the binding of the probe with its complementary strand showed a more intensified band than that of the reaction with Nb.BbvCI alone (compare lane 1 versus lane 3 in Figure 2), suggesting that the reaction with both Nb.BbvCI and Klenow produced ssDNA. In the absence of Nb.BbvCI, the hybrid band of the probe and its complementary strand was absent, proving the nickase activity of Nb.BbvCI. Based on these data, we concluded that Klenow possessed ssDNA replacing activity. However, in the reaction using Phi29, we somehow observed relatively weak band of the hybridized probe with its complementary strand in the system with both Nb.BbvCI and Phi29, compared to that with only the Nb.BbvCI (Figure 2). We are still investigating the underlying reason for this unexpected phenomenon.

Figure 3. 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.

We 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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