Difference between revisions of "Part:BBa K3790004"
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[[File:T--Fudan--ccic8-transparent-logo.png|100px|right|2021 Fudan]] | [[File:T--Fudan--ccic8-transparent-logo.png|100px|right|2021 Fudan]] | ||
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__TOC__ | __TOC__ | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | + | AlbA1 is a double-stranded binding protein, which is close to Sso7d in length and structure<ref name="7kd">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</ref>. We speculate that albA1 may be related to Sso7d, and it may have a similar function to enhance DNA polymerase activity as Sso7d<ref name="cao">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. | |
| − | </ref>. The Bst | + | </ref>. |
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| + | 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 albA1 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 and designed synthetic primers for synthesis | + | Since the length of the albA1 fragment is less than 500bp, we chose to synthesize the sequence ourselves by Oligo assembly using Phanta polymerase. We obtained the sequence information from [https://www.ncbi.nlm.nih.gov/nuccore/NC_002754.1?report=fasta&from=816731&to=817024&strand=true NCBI] 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.]] | [[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.]] | ||
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The length of albA1 DNA was 288 bp, which is approximately 300 bp after adding homology arms to both ends for PCR cloning. We isolated the DNA of interest by gel extraction for subsequent reactions. | The length of albA1 DNA was 288 bp, which is approximately 300 bp after adding homology arms to both ends for PCR cloning. We isolated the DNA of interest by gel extraction for subsequent reactions. | ||
| − | [[File:T--Fudan--albA1-S.ssb-E.ssb.jpg|600px|thumb|none| '''Figure 2. Assembled DNA binding proteins, | + | [[File:T--Fudan--albA1-S.ssb-E.ssb.jpg|600px|thumb|none| '''Figure 2. Assembled DNA binding proteins, albA1, S.ssb, E.ssb.''' 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> | ||
<partinfo>BBa_K3790004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3790004 SequenceAndFeatures</partinfo> | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
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| − | === | + | |
| + | ===References=== | ||
<references /> | <references /> | ||
Latest revision as of 04:01, 22 October 2021
albA1
Usage and Biology
AlbA1 is a double-stranded binding protein, which is close to Sso7d in length and structure[1]. We speculate that albA1 may be related to Sso7d, and it may have a similar function to enhance DNA polymerase activity as Sso7d[2].
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[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 albA1 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 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 albA1 DNA was 288 bp, which is approximately 300 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, albA1, S.ssb, E.ssb. 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
- ↑ 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
- ↑ 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.
- ↑ 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.