Difference between revisions of "Part:BBa J61001:Experience"

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Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.
 
Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.
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To characterize this origin of replication (ori) we tested it in two different strains of <i>E. coli</td> BW 295427 (also known as WM3064) containing the pir gene and <i>E. coli</i> DH5α which does not contain the pri gene [Ye L., Hildebrand F., Dingemans J., Ballet S., Laus G., Matthijs S., … Cornelis P. (2014). Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram‐positive and Pseudomonas sp. pathogens. PLoS ONE, 9, e110038.]. To somehow quantify the strength of the ori in those strains, we transformed them with a plasmid containing ori R6K and measured the DNA concentration after plasmid preparation with a commercial kit (Nucleospin Macherey-Nagel).
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To choose a correct plasmid for transformation, we made a PCR of two plasmids that should contain ori R6K.
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The resulting picture of an 1,2 % agaraose gel of the PCR product is shown below.
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<img src="oriR6K_gel_bronze"/>
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<p><i>Figure 1: 1,2 % agarose gel (150 V, 50 W, 300 mA, 25 min) of PCR from template plasmid used for transformation of <i>E. coli</td> BW29427 and <i>E. coli</td> DH5α. 1 and 2 are from the same plasmid preparation which should contain oriR6K. The additional bands in sample 2 could come from genomic DNA from the plasmid preparation.</i></p>
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As the expected band was present in line 2, we further used this plasmid for transformation.
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Therefore, we used this plasmid containing the ori R6K and a gene for a kanamycin resistance and transformed it in competent <i>E. coli</td> BW29427 and <i>E. coli</td> DH5α [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Previously we used the same batch of competent <i>E. coli</td> DH5α for transformations to test their transformation capability.
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Bild einfügen von erfolgreich transformierten dh5α
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The shown transformation was made with the same protocol and batch of <i>E. coli</td> Dh5α as a positive control, as it contains ori pMB1 which is known to work in <i>E. coli</td> Dh5α. Transformation in <i>E. coli</td> BW29427 was successful and there was no growth of <i>E. coli</td> DH5α due to selection of untransformed cells on LB-Kan agar plates.
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Afterwards we made a plasmid preparation of transformed <i>E. coli</td> BW29427 to get quantitative data for plasmid concentrations. Therefore, for a plasmid preparation of four batches of <i>E. coli</td> BW29427 was performed. OD(600) of overnight cultures was adjusted to 2,55 and a commercial kit was used for plasmid preparation (Nucleospin Macherey-Nagel). Final DNA concentrations were measured via nano-drop. The resulting table is shown below:
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<td>Culture number</td>
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<td> DNA concentration [ng/μL]</td>
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</tr>
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<td>1</td>
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<td>12,4</td>
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</tr>
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<td>2</td>
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<td>14,6</td>
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<td>3</td>
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<td>13,9</td>
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</tr>
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<td>4</td>
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<td>14,5</td>
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</tr>
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</table>
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<i>Table 1: DNA concentrations [ng/μL] after plasmid preparation of transformed E. coli BW29427.</i>
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To compare this result, we prepped plasmids from DH5α containing ori pMB1, where we archieved DNA concentrations between 190 – 280 ng/μL from cultures normed to the same OD(600) of 2,55.
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Plasmid concentration is low compared to ori pMB1 but expected as the copy number of ori R6K is 15 – 20 and the copy number of ori pMB1 is 500 – 700 [Morgan, K. (2014, February 14). Plasmids 101: Origin of Replication. Retrieved from https://blog.addgene.org/plasmid-101-origin-of-replication], meaning ori R6K is not viable for amplification of plasmids but works if high copy numbers are not needed.
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Revision as of 20:23, 16 October 2019

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_J61001

User Reviews

UNIQ4946e1406adad0c9-partinfo-00000000-QINU

BW 295427 (also known as WM3064) containing the pir gene and E. coli DH5α which does not contain the pri gene [Ye L., Hildebrand F., Dingemans J., Ballet S., Laus G., Matthijs S., … Cornelis P. (2014). Draft genome sequence analysis of a Pseudomonas putida W15Oct28 strain with antagonistic activity to Gram‐positive and Pseudomonas sp. pathogens. PLoS ONE, 9, e110038.]. To somehow quantify the strength of the ori in those strains, we transformed them with a plasmid containing ori R6K and measured the DNA concentration after plasmid preparation with a commercial kit (Nucleospin Macherey-Nagel). To choose a correct plasmid for transformation, we made a PCR of two plasmids that should contain ori R6K. The resulting picture of an 1,2 % agaraose gel of the PCR product is shown below.

Figure 1: 1,2 % agarose gel (150 V, 50 W, 300 mA, 25 min) of PCR from template plasmid used for transformation of E. coli BW29427 and E. coli DH5α. 1 and 2 are from the same plasmid preparation which should contain oriR6K. The additional bands in sample 2 could come from genomic DNA from the plasmid preparation.

As the expected band was present in line 2, we further used this plasmid for transformation. Therefore, we used this plasmid containing the ori R6K and a gene for a kanamycin resistance and transformed it in competent E. coli BW29427 and E. coli DH5α [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Previously we used the same batch of competent E. coli DH5α for transformations to test their transformation capability. Bild einfügen von erfolgreich transformierten dh5α The shown transformation was made with the same protocol and batch of E. coli Dh5α as a positive control, as it contains ori pMB1 which is known to work in E. coli Dh5α. Transformation in E. coli BW29427 was successful and there was no growth of E. coli DH5α due to selection of untransformed cells on LB-Kan agar plates. Afterwards we made a plasmid preparation of transformed E. coli BW29427 to get quantitative data for plasmid concentrations. Therefore, for a plasmid preparation of four batches of E. coli BW29427 was performed. OD(600) of overnight cultures was adjusted to 2,55 and a commercial kit was used for plasmid preparation (Nucleospin Macherey-Nagel). Final DNA concentrations were measured via nano-drop. The resulting table is shown below:
••••

UNIPV-Pavia iGEM 2010

BBa_K300008 was used as a validation construct for this conditional replication origin, in order to test its capability to be propagated in pir+ or pir-116 strain and its inability to propagate in the other E. coli strains.

In particular, BBa_K300008 was cut with XbaI-SpeI and the insert was isolated and purified from a 1% agarose gel. Then, it was self-ligated to generate a Cm-resistant R6K plasmid).

BW25141 (BBa_K300984) and BW23474 (BBa_K300985) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (BBa_V1001), MC1061 (BBa_K300078) and MG1655 (BBa_V1000) were chosen as pir- strains.


All these strains were made competent following the commonly used CaCl2 method [Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Then, a vial of 100 ul of competent cells was transformed with 2-4 ng of:

  • no DNA (negative control);
  • a pSB*** series vector (positive control);
  • self-ligated BBa_K300008.

and plated on LB+Cm at 34 ug/ml for high-copy plasmids, Cm at 12.5 ug/ml for medium/low copy plasmids and for the negative control strains transformed with the R6K plasmids.


The colonies were counted in each plate and the transformation efficiency was estimated in [CFU/ug of DNA] as:

efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]

The results are shown here:

Strain Efficiency with no DNA Efficiency with pSB*** (positive control) Efficiency with the self-ligated BBa_K300008 (R6K plasmid)
BBa_K300084 0 10^5 10^5
BBa_K300085 0 10^6 10^6
BBa_V1001 0 10^8 0
BBa_K300078 0 10^6 0
BBa_V1000 0 10^5 0

These results show that BBa_J61001 replication origin can be only propagated in pir+ and pir-116 strains (BBa_K300084 and BBa_K300085), while the transformation of other strains with the R6K plasmid yielded no colonies after transformation.

Moreover, these results show that the R6K plasmid in pir+ and pir-116 strains was transformed with the same efficiency as the pSB*** positive control plasmid, demonstrating that the R6K origin doesn't give any handicap in plasmid transformation.


To characterize this origin of replication (ori) we tested it in two different strains of E. coli
Culture number DNA concentration [ng/μL]
1 12,4
2 14,6
3 13,9
4 14,5
Table 1: DNA concentrations [ng/μL] after plasmid preparation of transformed E. coli BW29427. To compare this result, we prepped plasmids from DH5α containing ori pMB1, where we archieved DNA concentrations between 190 – 280 ng/μL from cultures normed to the same OD(600) of 2,55. Plasmid concentration is low compared to ori pMB1 but expected as the copy number of ori R6K is 15 – 20 and the copy number of ori pMB1 is 500 – 700 [Morgan, K. (2014, February 14). Plasmids 101: Origin of Replication. Retrieved from https://blog.addgene.org/plasmid-101-origin-of-replication], meaning ori R6K is not viable for amplification of plasmids but works if high copy numbers are not needed.


UNIQ4946e1406adad0c9-partinfo-00000014-QINU