Difference between revisions of "Part:pSB4A5:Experience"

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<partinfo>BBa_K864001 AddReview 1</partinfo>
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<I>[[User:agynna]]</I>
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'''iGEM Team Uppsala University 2012'''
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'''Warning: This is not a low copy backbone.''' The copy number of a sibling, pSB4C5 plasmid, with the same I50042 ori, has been estimated by three methods and compared to the pSB3C5 (with p15A ori) and the pSB4C15. The results shows strong evidence of <partinfo>I50042</partinfo> ori having a significantly higher copy number than specified, comparable to or higher than the p15A ori.
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*Measurement of fluorescence by FACS flow cytometry
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* Measurement of plasmid yields from liquid cultures
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*Visual color development of colonies on plates
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'''Flow cytometry'''
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[[Image:RFPflurescens.png|300px|thumb|Relative fluorescence of red cassette (J04450) in different backbones in E coli MG1655, with and without IPTG induction (0.5 mM). Quadruplicates (+IPTG samples) or triplicates (-IPTG). Fluorescence in arbitrary units, not compareable between +IPTG and -IPTG. Error bars are standard deviation. ]]
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E coli MG1655 was transformed with plasmids of different backbones with the J04450 standard RFP cassette. Liquid cultures were analysed by fluorescence with a Fluorescence activated cell sorter (FACS), quantitivly measuring the fluorescence of individual cells. Due to the active native lacI repression system in the bacteria, the experiment was performed with and without IPTG for promoter induction.
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Read about [[Part:BBa_I50042:Experience|I50042]] for details and other measurments.
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'''Conclusions'''
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The results demonstrate that <partinfo>pSB4C5</partinfo> has a significantly higher copy number than specified, of the same magnitude as <partinfo>pSB3C5</partinfo>. We are confident to conclude that the copy number regulation of pSB4C5 is broken. This conclusion can be expanded to the other pSB4x5 backbones, since they all share the I50042 origin of replication. Casual observations also support this result.
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The classic pSB4C5, and most likely the whole pSB4x5 series, are not low copy backbones as specified in the registry. They should not be used as low copy backbones. A possible future use of the pSB4x5 series is as a middle copy backbone that is compatible with the existing pSB3x5 (with p15A ori), something that is certainly useful from a syntetic biology standpoint.
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<partinfo>BBa_I50042 AddReview 5</partinfo>
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<I>[[User:Rshetty|Reshma Shetty]]</I>
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[[Part:BBa_I50042|BBa_I50042]] is a functional replication origin.  Based on typical miniprep yields of plasmids with [[Part:BBa_I50042|BBa_I50042]], it appears to be a low copy plasmid.  However, the plasmid copy number has not been verified.
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Revision as of 15:05, 27 September 2012

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 pSB4A5

User Reviews

UNIQc7bec1cb1d0bcfc6-partinfo-00000000-QINU

•

User:agynna

iGEM Team Uppsala University 2012

Warning: This is not a low copy backbone. The copy number of a sibling, pSB4C5 plasmid, with the same I50042 ori, has been estimated by three methods and compared to the pSB3C5 (with p15A ori) and the pSB4C15. The results shows strong evidence of BBa_I50042 ori having a significantly higher copy number than specified, comparable to or higher than the p15A ori.

  • Measurement of fluorescence by FACS flow cytometry
  • Measurement of plasmid yields from liquid cultures
  • Visual color development of colonies on plates

Flow cytometry

Relative fluorescence of red cassette (J04450) in different backbones in E coli MG1655, with and without IPTG induction (0.5 mM). Quadruplicates (+IPTG samples) or triplicates (-IPTG). Fluorescence in arbitrary units, not compareable between +IPTG and -IPTG. Error bars are standard deviation.

E coli MG1655 was transformed with plasmids of different backbones with the J04450 standard RFP cassette. Liquid cultures were analysed by fluorescence with a Fluorescence activated cell sorter (FACS), quantitivly measuring the fluorescence of individual cells. Due to the active native lacI repression system in the bacteria, the experiment was performed with and without IPTG for promoter induction.

Read about I50042 for details and other measurments.

Conclusions

The results demonstrate that pSB4C5 has a significantly higher copy number than specified, of the same magnitude as pSB3C5. We are confident to conclude that the copy number regulation of pSB4C5 is broken. This conclusion can be expanded to the other pSB4x5 backbones, since they all share the I50042 origin of replication. Casual observations also support this result.

The classic pSB4C5, and most likely the whole pSB4x5 series, are not low copy backbones as specified in the registry. They should not be used as low copy backbones. A possible future use of the pSB4x5 series is as a middle copy backbone that is compatible with the existing pSB3x5 (with p15A ori), something that is certainly useful from a syntetic biology standpoint.

•••••

Reshma Shetty

BBa_I50042 is a functional replication origin. Based on typical miniprep yields of plasmids with BBa_I50042, it appears to be a low copy plasmid. However, the plasmid copy number has not been verified.

•••••

Reshma Shetty

pSB4A5 has not been tested but is expected to be fully functional as a BioBrick standard vector. When miniprepped pSB4A5-I52002 gives high miniprep yield similar to a high copy plasmids.

•

User:Swanson

***Warning:***pSB4A5 does not appear to be correct in many distribution wells. The [http://2011.igem.org/Team:Washington 2011 Washington iGEM team] sequenced pSB4A5 from distributions in the past several years and found it to be in fact pSB1A2 - a high-copy, not a low-copy, plasmid. The UW iGEM team constructed pGA4A5, similar to pSB4A5 but for Gibson-friendly cloning. For more information, visit [http://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors Gibson Tookit] section of the Washington 2011 iGEM team's wiki.

UNIQc7bec1cb1d0bcfc6-partinfo-00000008-QINU