Coding
DbpA

Part:BBa_K3790001

Designed by: Guanqiao Chi   Group: iGEM21_Fudan   (2021-10-01)


DbpA


Introduction

2021 Fudan

DbpA is a double-stranded binding protein from Sulfolobus solfataricus (the same species as where Sso7d from), which is close to Sso7d in length and structure. We speculate that it may have a similar function to enhance DNA polymerase activity as Sso7d.

Usage and Biology

Sso7d is a double-stranded binding protein that is linked to DNA polymerase A or DNA polymerase B to produce a fusion protein with higher synthetic efficiency compared to wild-type DNA polymerase.

DbpA is a double-stranded binding protein from the same species as Sso7d, Sulfolobus solfataricus, which is close to Sso7d in length and structure[1]. We speculate that DbpA may be related to Sso7d, and it may have a similar function to enhance DNA polymerase activity as Sso7d[2].

The Bst Pol selected for this experiment was DNA polymerase Ⅰ, and no previous studies have focused on whether double-stranded binding proteins can enhance the activity of DNA polymerase Ⅰ[3]. However, we ventured to guess that fusing a double-stranded binding protein could enhance the related activity of DNA polymerase Ⅰ, and performed the following experiments.

Experimental Results

Since the length of the DbpA fragment is less than 500bp, we chose to synthesize the sequence ourselves by Oligo assembly using Phanta polymerase. We obtained the sequence information from NCBI[1] and designed synthetic primers for synthesis.

Figure 1. Oligo assembly by PCR. It is generally used to construct completely new or special-purpose DNA. This method may have the disadvantage of a high mutation rate when operated. Once, we had to sequence nine clones of the same construct to get a single correct one. The reason for this is most likely due to complex annealing and amplification. We suggest to have 10-15 rounds amplification without F1 or R1 primer, then add those two primers to have another 25 rounds. Must use high-fidelity enzymes for this method. Due to the pricing, we always use 60bp primers, 58 overlapping annealing temperature, to assemble 300-500bp DNA fragment.

The length of DbpA DNA is 219 bp, which is approximately 240 bp after adding homology arms to both ends for PCR cloning. We isolated the DNA of interest by gel extraction for subsequent reactions.

Figure 2. Assembled DNA binding proteins, DbpA, Sso10b, Sso7d. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures and sent for Sanger sequencing. Then, we verified the sequencing results, and used the correct ones for further experiments.


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]


References

  1. ↑ Kalichuk, V., Béhar, G., Renodon-Cornière, A. et al. The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family. Sci Rep 6, 37274 (2016). https://doi.org/10.1038/srep37274
  2. ↑ Cao S-C, Qiu L-Z. Study of DNA binding protein DbpA affecting the performance of DNA polymerase[J]. Journal of Fudan:Natural Science Edition, 2015, 54(4):469-477.
  3. ↑ Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res. 2004 Feb 18;32(3):1197-207. doi: 10.1093/nar/gkh271. PMID: 14973201; PMCID: PMC373405.
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