Building a Supercoilometer in the lab

Aim

To prepare a BioBrick from the tyrT promoter cloned from the E. coli genome and then construct and characterise a device which can detect the level of negative supercoiling in a closed circular plasmid.

Parts used

Plan for preparing the BioBricks:

1. Design suitable primers for cloning out the tyrT promoter sequence from the E. coli genome and have them synthesised from Eurofins MWG Operon. The primer design will need to include the necessary BioBrick prefixes and suffixes for standardisation of the cloned part
2. Carry out polymerase chain reaction (PCR) using the synthesised primers and Phusion polymerase to clone out the 103 bp tyrT promoter from the E. coli genome
3. Perform purification of the PCR products and prepare them into 50 μl samples
4. Carry out digestion of a sample of the PCR cloned tyrT promoter with EcoRI and SpeI

and another sample with EcoRI and PstI

1. Carry out digestion of linearised pSB1C3 plasmid backbone with EcoRI and PstI 6) Transform a stock of competent TOP10 E. coli cells with the CFP expression cassette

from well 8M on iGEM spring distribution - kit plate 1. Plate the cells on ampicillin plate, followed by an overnight culture in selective LB medium and finally prepare a 50 μl mini-prep sample

1. Carry out digestion of the CFP expression cassette with XbaI and PstI
2. Perform gel electrophoresis with a sample from the above digestion mixtures to

confirm the parts

1. Carry out ligation of the digested tyrT promoter (E/S), CFP expression cassette and

the pSB1C3 plasmid backbone. Also carry out a separate ligation of the digested tyrT promoter (E/P) and the digested pSB1C3 plasmid backbone

1. Transform separate stocks of competent TOP10 E. coli cells with the ligation mixtures

and grow them overnight on chloramphenicol plates

1. Set up overnight cultures from a single colony from the antibiotic plate in selective LB

medium

1. Set up a glycerol stock from each overnight culture (for storage at -80 °C) and mini-

prep the rest to extract the plasmids with the new BioBricks for the tyrT promoter and the supercoilometer device into 50 μl samples

1. Carry out digestion of the mini-prep samples with EcoRI and SpeI and perform gel

electrophoresis to confirm the length of the new BioBricks

1. Send a sample of the mini-prep DNA for sequencing to confirm the BioBricks

Barts design submitted to the Registry