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

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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===User Reviews===
 
===User Reviews===
 
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<partinfo>BBa_J61001 AddReview number</partinfo>
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<partinfo>BBa_J61001 AddReview 4</partinfo>
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<I>Username</I>
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<I>UNIPV-Pavia iGEM 2010</I>
 
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Enter the review inofrmation here.
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<partinfo>BBa_K300008</partinfo> 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.
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In particular, <partinfo>BBa_K300008</partinfo> 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).
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BW25141 (<partinfo>BBa_K3000984</partinfo>) and BW23474 (<partinfo>BBa_K3000985</partinfo>) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (<partinfo>BBa_V1001</partinfo>) and MC1061 (<partinfo>BBa_K3000978</partinfo>) were chosen as pir- strains.
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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);
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*self-ligated <partinfo>BBa_K300008</partinfo>.
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and plated on LB+Cm at 34 ug/ml.
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The colonies were counted in each plate and the transformation efficiency was estimated in '''[CFU/ug of DNA]''' as:
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efficiency [CFU/ug of DNA]= # CFU * 1000 ng of DNA / amount of transformed DNA [ng]
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The results are shown here:
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{|border=1
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|'''Strain'''
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|'''Efficiency with no DNA'''
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|'''Efficiency with pSB*** (positive control)'''
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|'''Efficiency with the self-ligated <partinfo>BBa_K300008</partinfo> (R6K plasmid)'''
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|-
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|<partinfo>BBa_K300084</partinfo>
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|0
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|10^5
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|10^5
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|-
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|<partinfo>BBa_K300085</partinfo>
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|0
 +
|10^6
 +
|10^6
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|-
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|<partinfo>BBa_V1001</partinfo>
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|0
 +
|10^8
 +
|0
 +
|-
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|<partinfo>BBa_K300078</partinfo>
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|0
 +
|10^6
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|0
 +
|}
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These results show that <partinfo>BBa_J61001</partinfo> replication origin can be only propagated in pir+ or pir-116 strains (<partinfo>BBa_K300084</partinfo> and <partinfo>BBa_K300085</partinfo>), while the transformation of other strains with the R6K plasmid yielded no colonies after transformation.
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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.
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|}
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Revision as of 17:22, 19 October 2010

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

UNIQ969ddef7655116d1-partinfo-00000000-QINU

••••

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 (No part name specified with partinfo tag.) and BW23474 (No part name specified with partinfo tag.) were chosen as pir+ and pir-116 strains respectively, while DH5alpha (BBa_V1001) and MC1061 (No part name specified with partinfo tag.) 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.


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

These results show that BBa_J61001 replication origin can be only propagated in pir+ or 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.

UNIQ969ddef7655116d1-partinfo-00000011-QINU