Part:pSB1C3: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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No review score entered. Genspace 2016

Plasmid Copy Number at Stationary Phase: 24 The Genspace 2016 iGEM team measured the copy number for pSB1C3 in Top10 both by qPCR and gel electrophoresis of lysate generated from cells harboring K909006-pSB1C3 and found the copy number to be much lower than indicated on the main page. qPCR suggested copy number was around 24 copies per cell and lysate electrophoresis indicated copy number was close to that detected by qPCR.

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••••• Waterloo iGEM 2014

The pSB1C3 backbone has been made more versatile by turning it into a shuttle vector. By cloning a Staphylococcal selective marker (erythromycin resistance gene) and origin of replication, this part has been improved to be used in both E. coli and Staphylococcal organisms. The Staphylococcal parts have been incorporated into the backbone between the VR and the pMB1 replication origin in pSB1C3. The newly integrated parts have been sequence confirmed and a restriction digest of this part isolated from E. coli shows expected bands: The shuttle vector was successfully electroporated and maintained in Staphylococcus epidermidis (ATCC 12228) and the newly transformed cell displayed erythromycin resistance. Whether or not the shuttle plasmid confers chloramphenicol resistance due to the E. coli gene is still inconclusive due to the fact that S. epidermidis was shown to have some resistance to chloramphenicol already. For more information, view the part page for this shuttle vector: BBa_K1323017

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••••• UNIPV-Pavia iGEM 2011

This cloning vector has been improved, cloning in it the strong pTet promoter, between E and X restriction sites; the mRFP coding sequence from BBa_J61002, has been placed between S and P restirction sites, in order to facilitate the cloning of coding sequences downstream pTet promoter in this high copy number plasmid. For more details see the BBa_K516999 experience page.

The 2011 Nevada iGEM Team attempted to use pSB1C3 as a template to amplify the chloramphenicol resistance cassette for use in a new part. When they attempted to design primers for this purpose, we were unable to identify a start codon. We subsequently contacted Dr. Knight and Austin Che about the problem. They re-examined the sequence and found that the sequence in the registry was missing two nucleotides which resulted in an apparent frameshift. Upon fixing the sequence annotation, we were able to design our amplification primers for the chloramphenicol cassette.

iGEM JHU Wetware 2012 The pSB1C3 backbone was utilized to create a new plasmid backbone specifically engineered for use with the Golden Gate assembly method. Flanking regions were added within the prefix and suffix that facilitate assembly through the use of Bsa1 overhangs. This makes for a scarless ligation into the vector. The resulting sequence is a standard pSB1C3 with a part of choice, as the overhangs are removed during the Bsa1 cleavage. Three varieties of the backbone were made.

• Part:BBa_K799024: pSB1C3 ProAV for promoters
• Part:BBa_K799025: pSB1C3 orfAV for coding regions
• Part:BBa_K799026: pSB1C3 ttAV for terminators

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