Difference between revisions of "Part:BBa K3790002"
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===Usage and Biology=== | ===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. | ||
| + | The Bst 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<ref name="novel">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.</ref>. 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=== | ===Experimental Results=== | ||
| + | Since the length of the Sso7d 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[https://www.ncbi.nlm.nih.gov/gene/?term=sso7d] and designed synthetic primers for synthesis. | ||
| + | [[File:T--Fudan--Oligo assembly by Taq polymerase.jpg|thumb|none|400px| '''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 Sso7d DNA was 174 bp, which is approximately 200 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA by gel extraction for subsequent reactions. | |
| + | [[File:T--Fudan--DbpA-sso10b-sso7d.jpg|600px|thumb|none| '''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.]] | ||
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| − | <span class='h3bb'>Sequence and Features</span> | + | <span class='h3bb'>'''Sequence and Features'''</span> |
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<partinfo>BBa_K3790002 parameters</partinfo> | <partinfo>BBa_K3790002 parameters</partinfo> | ||
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| + | ===References=== | ||
| + | <references /> | ||
Latest revision as of 17:35, 21 October 2021
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.
The Bst 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[1]. 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 Sso7d 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 Sso7d DNA was 174 bp, which is approximately 200 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA 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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ 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.