Part:pSB3T5:Experience

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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.

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

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

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.

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

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•••• Reshma Shetty

pSB3T5 is a functional low to medium copy plasmid but does not propagate at high copy. pSB3T5-I52001 has been used to successfully assemble BioBrick parts. However, pSB3T5-I52001 does not give high miniprep yields due to mutations in BBa_I52001 that render it nonfunctional as a high copy origin. pSB3T5-I52001 works fine as a plasmid but is not as convenient for plasmid DNA purification.

•••• Aberdeen_Scotland 2009

Plasmid rescued contained BBa_J04450. Transformation, miniprep, double and single digests all worked properly. It was noticeable that colonies were a light pink colour. We used this part for further cloning. This plasmid gave us a very high efficiency of cloning.

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