Difference between revisions of "Part:pSB3C5:Experience"

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===Applications of pSB3C5===
 
===Applications of pSB3C5===
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== '''Team Warwick 2015''' ==
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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.
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</p>
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<p>
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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.
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https://static.igem.org/mediawiki/2015/8/80/Warwick_J04450_Characterisation_graph.png
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<p>
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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.
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</p>
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<p>
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The raw data for this characterisation can be found here:
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<br> https://static.igem.org/mediawiki/2015/7/70/Warwick_J04450_raw_data.txt
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<br> https://static.igem.org/mediawiki/2015/b/bc/Warwick_J04450_in_pSB1K3_analysed_results.txt
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<br> https://static.igem.org/mediawiki/2015/8/8f/Warwick_J04450_in_pSB3K3_analysed_results.txt
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<br> https://static.igem.org/mediawiki/2015/5/54/Warwick_J04450_in_pSB4K5_analysed_results.txt
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<br> https://static.igem.org/mediawiki/2015/6/64/Warwick_J04450_RFP_OD_analysed_results.txt
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</p>
  
 
===User Reviews===
 
===User Reviews===

Revision as of 09:57, 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 pSB3C5

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

UNIQ9bf28323e5c7b9ac-partinfo-00000000-QINU

Ilya Vainberg Slutskin

pSB3C5, obtained from 2012 distribution kit well 3C, has consistently gave higher miniprep concentrations in comparison to pSB1C3. Even after taking into account the difference in plasmid size (in bp) the calculated copy number is higher for pSB3C5 than for pSB1C3. This suggests that this plasmid might not function as a medium-low copy plasmid. Similar observations have been found by Reshma Shetty for pSB3K5, which uses the same replication origin.

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Reshma Shetty

pSB3C5 is a functional low to medium copy plasmid but does not propagate at high copy. When transformed into TOP10 cells, pSB3C5-I52001 does not transform as expected. When pSB3C5-I52001 is cut with NotI, self-ligated (to eliminate ccdB positive selection marker) and tranformed into TOP10, lots of colonies are obtained. However, pSB3C5-I52001 does not give high miniprep yields due to mutations in BBa_I52001 that render it nonfunctional as a high copy origin. pSB3C5-I52001 works fine as a plasmid but is not as convenient for plasmid DNA purification.

Aberdeen_Scotland 2009

The transformation was twice unsuccessful. Plasmid rescued contained BBa_J04450.

UNIQ9bf28323e5c7b9ac-partinfo-00000004-QINU