Experiments
taramaramaramparam
Cloning
Cloning was performed using Biobrick Standard Assembly method. All cloning procedures were carried out in pSB1C3 vector.
Firstly, plasmids, containing three promoters and GFP gene, were transformed into E. coli DH5α strain. Bacteria, containing the plasmids, were grown overnight and then DNA extraction ensued. The extracted DNA was cut using EcoRI and SpeI restriction enzymes for the promoters, and with EcoRI and XbaI for plasmids, containing the GFP gene. Later, plasmids with incorporated GFP gene were purified using PCR cleaning kit and the promoters were extracted from gel using freeze and squeeze protocol. Then the promoters were ligated into vectors, containing GFP gene, ligation products were again transformed into E. coli DH5α strain. The validity of our constructs was tested by restriction mapping – using StyI restrictase.
Growing
All three constructs were grown as three biological replicates. We used positive control – a GFP expressing device – BBa_I20270. We also used a negative control, which did not have the GFP gene in it – BBa_R0040. The positive and negative controls were grown in biological triplicates. All bacteria were grown in a LB medium overnight.