Difference between revisions of "Part:BBa K1932001"
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Part BBa_K1932001was originally cloned from the plasmid of Bifidobacterium that contains two orf sequences.（The plasmid had a G + C content of 62.0%, and contained two open reading frames, orf1 and orf2）.This sequence is essential for the shuttling of the plasmid from E.<i>coli</i> to Bifidobacterium.
 
Part BBa_K1932001was originally cloned from the plasmid of Bifidobacterium that contains two orf sequences.（The plasmid had a G + C content of 62.0%, and contained two open reading frames, orf1 and orf2）.This sequence is essential for the shuttling of the plasmid from E.<i>coli</i> to Bifidobacterium.
  
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Characterization:
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<p style="font-size:130%">'''Characterization:'''</p>
  
 
The part of BBa_K1932001 was synthesized and cloned into a pGH vector by Generay Biotechnology. This plasmid was cut with the restriction enzyme, EcoRⅠand PstⅠ, and separated by 1% agarose gel (Fig.1).  
 
The part of BBa_K1932001 was synthesized and cloned into a pGH vector by Generay Biotechnology. This plasmid was cut with the restriction enzyme, EcoRⅠand PstⅠ, and separated by 1% agarose gel (Fig.1).  
 +
 +
"https://static.igem.org/mediawiki/2016/d/da/T--Jilin_China--p1-1.png"
 +
 +
<p style="font-size:75%">'''Fig.1. (1): pGH+PMB1; (2) marker;3:pGH+PMB1 digested with EcoRⅠ and PSTⅠ'''</p>
  
 
The sequence of PMB1 was ligated into the vector pSB1C3 by ligase in 16℃ overnight, and the ligated products were transformed into the E.<i>coli</i>(Fig.2).
 
The sequence of PMB1 was ligated into the vector pSB1C3 by ligase in 16℃ overnight, and the ligated products were transformed into the E.<i>coli</i>(Fig.2).
  
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To ensure the insertion of the right-size sequence, the sequence of PMB1 was cut again and tested by agarose gel electrophoresis (Fig.3).  
+
"https://static.igem.org/mediawiki/2016/a/a3/T--Jilin_China--p1-2.png"
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<p style="font-size:75%">'''Fig.2. (1) control only DH5α; (2) DH5α transformed with BBa_K1932001 (the PMB1+pSB1C3 vector)'''</p>
  
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Once the size of this sequence was confirmed, the bacteria containing the construct were sent to the Comate Bioscience Company for DNA sequencing for further verification. The detailed protocols of these experiments were shown in table 1 and table 2.
 
  
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The sequence of PMB1 differs from the sequence of pMB1 from the vector, pSB1C3,which was shown with the tool of BLAST online(Fig.4).
+
The detailed protocols of these experiments were shown in table 1 and table 2.
  
 +
The sequence of PMB1 differs from the sequence of pMB1 from the vector, pSB1C3,which was shown with the tool of BLAST online(Fig.3).
  
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Reference
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<p style="font-size:130%">'''References:'''</p>
  
 
【1】Rossi, M., Brigidi, P., y Rodriguez, A. G. V., &Matteuzzi, D. (1996).Characterization of the plasmid pMB1 from Bifidobacterium <i>longum</i> and its use for shuttle vector construction.<i>Research in microbiology, 147(3)</i>, 133-143.
 
【1】Rossi, M., Brigidi, P., y Rodriguez, A. G. V., &Matteuzzi, D. (1996).Characterization of the plasmid pMB1 from Bifidobacterium <i>longum</i> and its use for shuttle vector construction.<i>Research in microbiology, 147(3)</i>, 133-143.

Revision as of 19:31, 19 October 2016


orf1 and orf2 from Bifidobacterium

Part BBa_K1932001was originally cloned from the plasmid of Bifidobacterium that contains two orf sequences.（The plasmid had a G + C content of 62.0%, and contained two open reading frames, orf1 and orf2）.This sequence is essential for the shuttling of the plasmid from E.coli to Bifidobacterium.

Characterization:

The part of BBa_K1932001 was synthesized and cloned into a pGH vector by Generay Biotechnology. This plasmid was cut with the restriction enzyme, EcoRⅠand PstⅠ, and separated by 1% agarose gel (Fig.1).

"T--Jilin_China--p1-1.png"

Fig.1. (1): pGH+PMB1; (2) marker;3:pGH+PMB1 digested with EcoRⅠ and PSTⅠ

The sequence of PMB1 was ligated into the vector pSB1C3 by ligase in 16℃ overnight, and the ligated products were transformed into the E.coli(Fig.2).

"T--Jilin_China--p1-2.png"

Fig.2. (1) control only DH5α; (2) DH5α transformed with BBa_K1932001 (the PMB1+pSB1C3 vector)


The detailed protocols of these experiments were shown in table 1 and table 2.

The sequence of PMB1 differs from the sequence of pMB1 from the vector, pSB1C3,which was shown with the tool of BLAST online(Fig.3).

References:

【1】Rossi, M., Brigidi, P., y Rodriguez, A. G. V., &Matteuzzi, D. (1996).Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction.Research in microbiology, 147(3), 133-143.

【2】Matteuzzi, D.,Brigidi, P., Rossi, M., & Di, D. (1990).Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1.Letters in applied microbiology, 11(4), 220-223.

【3】Xu, Y. F., Zhu, L. P., Hu, B., Fu, G. F., Zhang, H. Y., Wang, J. J., &Xu, G. X. (2007). A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy.Cancer gene therapy, 14(2), 151-157.

【4】Corneau, N., Émond, É., &LaPointe, G. (2004).Molecular characterization of three plasmids from Bifidobacterium longum.Plasmid, 51(2), 87-100.

【5】Hu, B., Kou, L., Li, C., Zhu, L. P., Fan, Y. R., Wu, Z. W., ... &Xu, G. X. (2009). Bifidobacterium longum as a delivery system of TRAIL and endostatin cooperates with chemotherapeutic drugs to inhibit hypoxic tumor growth. Cancer gene therapy, 16(8), 655-663.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 935