Difference between revisions of "Part:BBa K3790006"

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===Introduction===
 
 
[[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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E.ssb is a single-stranded binding protein from Escherichia coli str. K-12 substr. MG1655[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=511145]. We wanted to test whether the single-stranded binding protein could enhance the enzymatic activity of Bst Pol using the change in the activity of the fusion protein composed of it and Bst Pol.
 
  
 
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===Usage and Biology===
 
===Usage and Biology===
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In past studies, double-stranded binding proteins, represented by sso7d, were often thought to enhance the activity of DNA polymerase A or DNA polymerase B. However, as a single-stranded binding protein that can also bind DNA, there is no study to prove whether it can increase the activity of DNA polymerase. Therefore, we chose E.ssb, a single-stranded binding protein from scherichia coli str. K-12 substr. MG1655 to verify whether the single-stranded binding protein could enhance the enzymatic activity of Bst Pol.
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In past studies, double-stranded binding proteins, represented by Sso7d, were shown by our lab and others to enhance the activity of DNA polymerase A or DNA polymerase B<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>. However, as a single-stranded binding protein that can also bind DNA, there is no study to prove whether it can increase the activity of DNA polymerase. Therefore, we chose E.ssb, a single-stranded binding protein from ''Escherichia coli'' [https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=511145 str. K-12 substr. MG1655] to test whether the single-stranded binding protein could enhance the enzymatic activity of Bst.
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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<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>.
  
 
===Experimental Results===
 
===Experimental Results===
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Since the length of the E.ssb fragment is less than 600bp, 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/948570] and designed synthetic primers for synthesis.
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Since the length of the E.ssb fragment is less than 600bp, 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/gene/948570 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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[[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.]]
 
[[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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===Reference===
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>'''Sequence and Features'''</span>
 
<partinfo>BBa_K3790006 SequenceAndFeatures</partinfo>
 
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<partinfo>BBa_K3790006 parameters</partinfo>
 
<partinfo>BBa_K3790006 parameters</partinfo>
 
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===References===
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<references />

Latest revision as of 17:54, 21 October 2021


E.ssb


2021 Fudan


Usage and Biology

In past studies, double-stranded binding proteins, represented by Sso7d, were shown by our lab and others to enhance the activity of DNA polymerase A or DNA polymerase B[1]. However, as a single-stranded binding protein that can also bind DNA, there is no study to prove whether it can increase the activity of DNA polymerase. Therefore, we chose E.ssb, a single-stranded binding protein from Escherichia coli str. K-12 substr. MG1655 to test whether the single-stranded binding protein could enhance the enzymatic activity of Bst.

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[2].

Experimental Results

Since the length of the E.ssb fragment is less than 600bp, 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 E.ssb DNA was 531 bp, which is approximately 550 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:
  • 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. ↑ 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.
  2. ↑ 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.