Difference between revisions of "Part:pSB4A5:Experience"

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===Applications of pSB4A5===
 
===Applications of pSB4A5===
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== '''Team Warwick 2016''' ==
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 +
This year, our team demonstrated that this part can be triple transformed into the same competent cell while retaining the functionality of all the plasmids. This was demonstrated using pSB1C3, pSB3T5, and pSB4A5.
 +
 +
pSB4A5 is a BioBrick standard vector with a pSC101 replication origin, whilst pSB3T5 has a p15A replication origin. As both of these origins rely on iterons as the negative copy number control, the competition between the plasmids may render them incompatible when in the same cell[1], potentially limiting triple transformation efficiency. However through our experiment we show that the replication origins do not interfere with each other.
 +
 +
To illustrate this, we transferred the triple transformed cells onto plates containing chloramphenicol, tetracycline, and ampicillin, as shown in Figure 1 below. From this plate, we inoculated a single colony into water and streaked this water onto one half of three different plates containing one each of chloramphenicol, tetracycline, and ampicillin. On the other half of the plates, we streaked a colony of top10 cells that had been grown on a streptomycin plate. The results can be seen below in figures 2 and 3. Figure 3 shows the four plates under UV light, clearly showing where colonies have grown. As the triple transformed cells have grown on every single plates and no colonies have grown from the untransformed cells, this shows that plasmid functionality is retained.
 +
 +
https://static.igem.org/mediawiki/2016/8/84/T--Warwick--ImprovImage1.jpg
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Figure 1: Visible light image of triple transformation plated on Chlor-Amp-Tet plate
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 +
https://static.igem.org/mediawiki/2016/1/10/T--Warwick--ImprovImage2.jpg
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 +
Figure 2: Visible light image of triple transformation plated on Chlor, Amp, and Tet plates alongside untransformed Top10 cells
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 +
https://static.igem.org/mediawiki/2016/2/28/T--Warwick--ImprovImage3.jpg
 +
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Figure 3: UV light image of triple transformation plated on Chlor, Amp, and Tet plates alongside untransformed Top10 clearly showing differences in growth
 +
 +
== '''Team Warwick 2015''' ==
 +
 +
<p>
 +
Our team considered using this part as part of a system of binding different coloured cells together in order to demonstrate specific cell placement. We characterised this part is order to determine the optimal amount of IPTG required for inducing the gene, and the copy number necessary to express the fluorescence brightly. The results for this can be seen below.
 +
</p>
 +
 +
<p>
 +
We cloned J04450 into three plasmid with varying copy numbers, in order from highest to lowest copy number they are: pSB1K3, pSB3K3, and pSB4K5. These plasmids were then transformed into electrocompetent MG1655 Z1 cells and grown overnight. THe next morning the cells were refreshed, and different concentrations of IPTG (0uM, 250uM, and 500uM) were added to induce them. For each of the three plasmids in each IPTG concentrations, three biological replicates were made, and when OD600 and RFP absorbance were measured, three technical replicates were made, for a total of 81 copies of the gene grown. The RFP absorbance and OD600 of these cells were measured over 20 hours. The OD600 over time was used to determine at what OD the cells were in steady state. This was then compared to the RFP measured at that time and graphed to show RFP expression per cell.
 +
</p>
 +
 +
https://static.igem.org/mediawiki/2015/8/80/Warwick_J04450_Characterisation_graph.png
 +
 +
<p>
 +
The graph shows that RFP expression was highest in the pSB1K3 and pSB4K5 plasmids, and that there was little difference in expression between the 250uM and 500uM concentration of IPTG. 0uM IPTG universally showed almost no expression. pSB1K3 should have the highest copy number and pSB4K5 should have the lowest copy number, so it's curious that they both expressed RFP very well. This could be due to a mutation in the pSB4K5 causing it to have a much higher copy number than usual. It is documented  here (http://www.ncbi.nlm.nih.gov/pubmed/1283002) that a single point mutation can increase the copy number of a plasmid. The pSB4K5 plasmid we tested has been sent for sequencing in order to determine whether this is the case.
 +
</p>
 +
 +
<p>
 +
The raw data for this characterisation can be found here:
 +
<br> https://static.igem.org/mediawiki/2015/7/70/Warwick_J04450_raw_data.txt
 +
<br> https://static.igem.org/mediawiki/2015/b/bc/Warwick_J04450_in_pSB1K3_analysed_results.txt
 +
<br> https://static.igem.org/mediawiki/2015/8/8f/Warwick_J04450_in_pSB3K3_analysed_results.txt
 +
<br> https://static.igem.org/mediawiki/2015/5/54/Warwick_J04450_in_pSB4K5_analysed_results.txt
 +
<br> https://static.igem.org/mediawiki/2015/6/64/Warwick_J04450_RFP_OD_analysed_results.txt
 +
</p>
 +
  
 
===User Reviews===
 
===User Reviews===
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|-
 
|width='10%'|
 
|width='10%'|
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<partinfo>pSB4A5 AddReview number</partinfo>
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<partinfo>pSB4A5 AddReview 1</partinfo>
 
<I>Username</I>
 
<I>Username</I>
 
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|width='60%' valign='top'|
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|};
 
|};
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_K864001 AddReview 1</partinfo>
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<I>[[User:agynna]]</I>
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|width='60%' valign='top'|
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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.
 +
 +
*Measurement of fluorescence by FACS flow cytometry
 +
* Measurement of plasmid yields from liquid cultures
 +
*Visual color development of colonies on plates
 +
 +
'''Flow cytometry'''
 +
 +
[[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. ]]
 +
 +
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 [[Part:BBa_I50042:Experience|I50042]] for details and other measurments.
 +
 +
'''Conclusions'''
 +
 +
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.
 +
 +
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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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_I50042 AddReview 5</partinfo>
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<I>[[User:Rshetty|Reshma Shetty]]</I>
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|width='60%' valign='top'|
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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.
 +
|}
  
 
{|width='80%' style='border:1px solid gray'
 
{|width='80%' style='border:1px solid gray'
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<I>[[User:Swanson]]</I>
 
<I>[[User:Swanson]]</I>
 
|width='60%' valign='top'|
 
|width='60%' valign='top'|
−
'''***Warning:***'''[[Part:pSB4A5|pSB4A5]] does not appear to be correct in any 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.
+
'''***Warning:***'''[[Part:pSB4A5|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.
 
|}
 
|}
 
<!-- DON'T DELETE --><partinfo>pSB4A5 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>pSB4A5 EndReviews</partinfo>

Latest revision as of 09:56, 24 October 2016

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

Team Warwick 2016

This year, our team demonstrated that this part can be triple transformed into the same competent cell while retaining the functionality of all the plasmids. This was demonstrated using pSB1C3, pSB3T5, and pSB4A5.

pSB4A5 is a BioBrick standard vector with a pSC101 replication origin, whilst pSB3T5 has a p15A replication origin. As both of these origins rely on iterons as the negative copy number control, the competition between the plasmids may render them incompatible when in the same cell[1], potentially limiting triple transformation efficiency. However through our experiment we show that the replication origins do not interfere with each other.

To illustrate this, we transferred the triple transformed cells onto plates containing chloramphenicol, tetracycline, and ampicillin, as shown in Figure 1 below. From this plate, we inoculated a single colony into water and streaked this water onto one half of three different plates containing one each of chloramphenicol, tetracycline, and ampicillin. On the other half of the plates, we streaked a colony of top10 cells that had been grown on a streptomycin plate. The results can be seen below in figures 2 and 3. Figure 3 shows the four plates under UV light, clearly showing where colonies have grown. As the triple transformed cells have grown on every single plates and no colonies have grown from the untransformed cells, this shows that plasmid functionality is retained.

T--Warwick--ImprovImage1.jpg

Figure 1: Visible light image of triple transformation plated on Chlor-Amp-Tet plate

T--Warwick--ImprovImage2.jpg

Figure 2: Visible light image of triple transformation plated on Chlor, Amp, and Tet plates alongside untransformed Top10 cells

T--Warwick--ImprovImage3.jpg

Figure 3: UV light image of triple transformation plated on Chlor, Amp, and Tet plates alongside untransformed Top10 clearly showing differences in growth

Team Warwick 2015

Our team considered using this part as part of a system of binding different coloured cells together in order to demonstrate specific cell placement. We characterised this part is order to determine the optimal amount of IPTG required for inducing the gene, and the copy number necessary to express the fluorescence brightly. The results for this can be seen below.

We cloned J04450 into three plasmid with varying copy numbers, in order from highest to lowest copy number they are: pSB1K3, pSB3K3, and pSB4K5. These plasmids were then transformed into electrocompetent MG1655 Z1 cells and grown overnight. THe next morning the cells were refreshed, and different concentrations of IPTG (0uM, 250uM, and 500uM) were added to induce them. For each of the three plasmids in each IPTG concentrations, three biological replicates were made, and when OD600 and RFP absorbance were measured, three technical replicates were made, for a total of 81 copies of the gene grown. The RFP absorbance and OD600 of these cells were measured over 20 hours. The OD600 over time was used to determine at what OD the cells were in steady state. This was then compared to the RFP measured at that time and graphed to show RFP expression per cell.

Warwick_J04450_Characterisation_graph.png

The graph shows that RFP expression was highest in the pSB1K3 and pSB4K5 plasmids, and that there was little difference in expression between the 250uM and 500uM concentration of IPTG. 0uM IPTG universally showed almost no expression. pSB1K3 should have the highest copy number and pSB4K5 should have the lowest copy number, so it's curious that they both expressed RFP very well. This could be due to a mutation in the pSB4K5 causing it to have a much higher copy number than usual. It is documented here (http://www.ncbi.nlm.nih.gov/pubmed/1283002) that a single point mutation can increase the copy number of a plasmid. The pSB4K5 plasmid we tested has been sent for sequencing in order to determine whether this is the case.

The raw data for this characterisation can be found here:
https://static.igem.org/mediawiki/2015/7/70/Warwick_J04450_raw_data.txt
https://static.igem.org/mediawiki/2015/b/bc/Warwick_J04450_in_pSB1K3_analysed_results.txt
https://static.igem.org/mediawiki/2015/8/8f/Warwick_J04450_in_pSB3K3_analysed_results.txt
https://static.igem.org/mediawiki/2015/5/54/Warwick_J04450_in_pSB4K5_analysed_results.txt
https://static.igem.org/mediawiki/2015/6/64/Warwick_J04450_RFP_OD_analysed_results.txt


User Reviews

UNIQ5de8bd7f084dfbc0-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.

UNIQ5de8bd7f084dfbc0-partinfo-00000008-QINU