Experiment

Building GBS BioBricks

Aim: To build three new BioBricks out of the gyrase binding sites (GBS) we have identified from literature and characterise their property

Parts used:

Procedure for preparing the BioBricks:

Designed suitable primers for cloning out the gyrase binding sites (GBS) from bacteriophage Mu genome, pSC101 and pBR322 plasmid and had them synthesised from Eurofins MWG Operon. The primer design included the necessary BioBrick prefixes and suffixes for standardisation of the cloned parts Carried out polymerase chain reaction (PCR) using our synthesised primers and Phusion polymerase to clone out the GBSs from the Mu genome, pSC101 and pBR322 plasmids Performed purification of the PCR products and prepared them into 50 μl samples Carried out digestion of the three PCR cloned GBS with EcoRI and PstI Carried out digestion of the mini-prep sample of the 2009 Spy device for detecting stress with XbaI and PstI Carried out digestion of linearised pSB1C3 and pSB1A3 plasmid backbones with EcoRI and PstI Performed gel electrophoresis with a sample from the six digestion mixtures stated above to confirm the parts Carried out ligation of each GBS separately with pSB1C3 plasmid backbone. Transformed separate stocks of competent TOP10 E. coli cells with the ligation mixtures and grew them overnight on chloramphenicol plates Set up overnight cultures from a single colony from the antibiotic plate in selective LB medium Set up a glycerol stock from each overnight culture (for storage at -80 °C) and mini-prepped the rest to extract the plasmids with the new BioBricks for the GBS into 50 μl samples Carried out digestion of the mini-prep samples with EcoRI and SpeI and performed gel electrophoresis to confirm the length of the new BioBricks Sent a sample of the mini-prep for sequencing to confirm the BioBrick Submitted the three new BioBricks to the registry (BBa_K676001; BBa_K676011; BBa_K676012) Carried out digestion of the mini-prep DNA for Mu GBS BioBrick with EcoRI and SpeI and performed gel electrophoresis of sample to confirm the digestion Carried out ligation with the digested Mu GBS, Spy device and pSB1A3 plasmid backbone to assemble a new composite BioBrick (BBa_K676013) Transformed a stock of competent TOP10 E. coli cell with the new composite BioBrick plasmid and plated the cell on ampicillin plates and set up overnight culture from a healthy colony in selective LB medium Set up a glycerol stock from the overnight culture (for storage at -80 °C) and mini-prepped the rest to extract the plasmid with the new composite BioBricks into a 50 μl sample Sent the mini-prep DNA for sequencing to confirm the composite BioBrick Submitted the new composite BioBrick to the registry (BBa_K676013)

Parts submitted to registry:

Part Name Parts ID Parts Description Length (bp) Plasmid Backbone Bacteriophage Mu GBS BBa_K676001 Gyrase Binding Site from Mu Bacteriophage genome 363 on pSB1C3 (2070 bp) - chloramphenicol resistance pSC101 GBS BBa_K676011 Gyrase Binding Site from pSC101 plasmid 333 on pSB1C3 (2070 bp) pBR322 GBS BBa_K676012 Gyrase Binding Site from pBR322 plasmid 326 on pSB1C3 (2070 bp) Mu GBS/Spy Device BBa_K676013 Mu GBS ligated upstream of the 2009 Spy device for measuring stress 1416 on pSB1A3 (2155 bp) - ampicillin resistance

Characterisation approach for comparing the quality of supercoiling induced by each GBS:

1. Transformed a stock of competent TOP10 E. coli cells with a pSB1C3 plasmid carrying a RFP coding device (from well 3A of iGEM 2011 Spring Distribution - Kit Plate 1)
2. Transformed separate stocks of competent TOP10 E. coli cells with the mini-prep samples prepared for the new GBS BioBricks on pSB1C3 plasmid backbones ligation mixtures and grew them overnight on antibiotic plates and then selective LB medium.
3. Set up four overnight shake flask cultures of the three GBS containing plasmids and a control pSB1C3 with RFP coding device in 50 LB medium in each culture
4. Placed the shake flask cultures in an incubator for growth at 37 °C and 175 rpm for 16 hours
5. Centrifuged the shake flask cultures to collect the cell pellets after overnight growth
6. Mini-prepped the cell pellets into 50 μl sample
7. Performed one dimensional chloroquine gel assay using the same amount of mini-prep DNA for all four cultures in order to compare the difference in the level and quality of supercoiling for each plasmid

Characterisation approach for comparing the stress response and supercoiling due to the presence and absence of Mu GBS on a plasmid

1. Transformed a stock of competent TOP10 E. coli cells with the Spy device for measuring stress response (BBa_K239009) from the UCL iGEM 2009 team and prepared glycerol stock
2. Set up two separate overnight culture from the glycerol stocks for the Spy device and the Mu GBS/Spy Device in 2 ml selective LB medium to prepare a seed stock
3. Measured the OD readings of the seed stocks in the morning and inoculated shake flasks containing 50 ml selective LB medium with approximately the same cell density in the inoculum
4. Placed the shake flasks in the incubator for growth at 37 °C and 175 rpm for 24 hours
5. Collected two 1 ml samples from each shake flask every 2 hours throughout the fermentation including one from time-point 0 to 24 hours
6. Measured the OD readings of the first sample instantly after they were collected and used the readings to plot a growth curve later on
7. Stored the second samples at -80 °C and after the fermentation, 200 μl from each sample were loaded onto a 96 well plate and used to measure the GFP fluorescence using #the Tecan Safire II microplate reader
8. Centrifuged the remaining volume of the shake flask fermentation in order to collect the cell pellet
9. Mini-prepped the cell pellets into separate 50 μl DNA samples
10. Performed one dimensional chloroquine gel assay using mini-prep DNA for Spy device and Mu GBS/Spy device plasmids in order to see the difference in the level of supercoiling