Data

Data for our favourite parts together with characterisation in Experience section:

1. BBa_K676001 - Mu-phage Gyrase Binding Site (GBS). We have shown that plasmids with the Mu GBS incorporated in them achieved a higher level of supercoiling with an improved uniformity or homogeneity. Data for the characterisation of this part is shown in the Experience section and is documented in the 2011 UCL iGEM wiki Magneto-sites Results section.
2. BBa_K676011 pSC101 GBS. We have shown that the presence pSC101 GBS in a plasmid results in a higher level of supercoiling compared to a control. However the extent of supercoiling achieved is less efficient compared to the plasmids incorporating Mu phage GBS, even though the gyrase binding affinity of pSC101 is reported to be higher than that of Mu-phage GBS in scientific literature. Data for the characterisation of this part is shown in the Experience section and is documented in the 2011 UCL iGEM wiki Magneto-sites Results section.
3. BBa_K676012 - pBR322 GBS. We have shown that the presence of pBR322 GBS in a plasmid results in a decrease in the level of supercoiling achieved when compared to a control plasmid with no GBS in it. Data for the characterisation of this part is shown in the Experience section and is documented in the 2011 UCL iGEM wiki Magneto-sites Results section.

Data for the existing parts designed by another team with characterisation:

1. BBa_J04450 RFP Coding Device - According to our result in the 1D chloroquine gel electrophoresis (at 2.5 μg/mL), the plasmid with the BBa_J04450 (on pSB1C3 backbone) migrated further compared to a plasmid with a known GBS (from pBR322 plasmid - BBa_K676012) on pSB1C3. At the chloroquine concentration which we used, the more negatively supercoiled plasmid will migrate further while the more relaxed molecules will travel slower through the gel. So this indicates that the device BBa J04450 might have a potential gyrase binding site which facilitates the introduction of negative supercoils into plasmid compared to pBR322 GBS.

Data for the improved parts with characterisation:

1. BBa_K676013 Mu-GBS(K676001) + SPY promoter(K239001) +RBS(B0034) +GFP(E0040)+Double terminator(B0010,B0012). We have shown that with this new constructed composite, we have achieved a higher expression level of GFP from the Spy promoter. In this way, we have made the GFP expression cassette more sensitive to shear stress, which is normally encountered during fermentation and downstream processing.

Data for parts under construction

1. BBa_K676004 - The TyrT promoter+RBS(B0034)+eCFP(E0022)+Double terminator(B0010,B0012). The transcription from the TyrT promoter is sensitive to the topological state of the plasmid inside a cell. Thus by measuring the level of expression of the eCFP from the expression cassette, we will be able to determine the level of supercoiling produced in the plasmid DNA in vivo.
2. BBa_K676006 The T5 phage exonuclease. The exonuclease will be expressed under an inducible promoter during the cell harvesting stage in downstream processing. By expressing the exonuclease, non-desirable plasmids topoisomers (those which are damaged or are not supercoiled) will be digested leaving the negatively supercoiled plasmid in their a pure state. This would then make the downstream processing to purify the negatively supercoiled target plasmids simpler, faster and more efficient.
3. BBa_K676008 The LacI promoter(R0010)+RBS(B0034)+Gyr A (K676007)+ double terminator(B0010,B0012). The transcription of Gyr A subunits is induced by the IPTG inducible promoter. Gyr A is the subunit involved in DNA cleavage as well as DNA resealing and is essential for the formation of a fully functional tetrameric (A2 B2) gyrase enzyme. Unfortunately due to the time constraint and some difficulties in PCR reactions we are unable to complete the construction and characterisation on time.
4. BBa_K676010 The LacI promoter(R0010)+RBS(B0034)+Gyr B (K676009)+ double terminator(B0010,B0012). The transcription of Gyr B subunits is induced by the IPTG inducible promoter. Gyr B contains the ATPase and is essential for the formation of a fully functional tetrameric (A2 B2) gyrase. Unfortunately due to the time constraint and some difficulties in PCR reactions we were unable to complete the construction and characterisation on time.