| - | An ncAA synthetase/tRNA pair was cloned into pBLG and transformed into pFPYC Amberless E. Coli. and pFPY Amberless E. Coli. Other necessary control strains include mCherry Amberless E.Coli (RFP control), sfGFP Amberless E. Coli (GFP control), Amberless E. Coli (OD 600 control), and LB with ncAA (media background). An overnight culture of each strain was grown in LB with the appropriate antibiotics at 37 degrees Celsius and 225rpm. 10 milliliters of media with the appropriate antibiotics was inoculated with 100 microliter of overnight culture and allowed to grown under the same environmental conditions until the culture density was 0.2~0.3 OD, approximately 3 hours. The 10 milliliter culture was split between 4 different sterile test-tubes, 2 milliliters of culture per tube. The conditions of test tubes A through D were as follows: A (-IPTG,-ncAA), B (-IPTG,+ncAA), C (+IPTG, -ncAA), and D (+IPTG, +ncAA). IPTG stock solution was made at 1000X concentration and the ncAA was added to yield 1 mM concentration. Sterile DI water was added in the place of ncAA and IPTG as a control. | + | An ncAA synthetase/tRNA pair was cloned into pBLG and transformed into pFPYC Amberless E. Coli. and pFPY Amberless E. Coli. Other necessary control strains include mCherry Amberless E.Coli (RFP control), sfGFP Amberless E. Coli (GFP control), Amberless E. Coli (OD 600 control), and LB with ncAA (media background). An overnight culture of each strain was grown in LB with the appropriate antibiotics at 37 degrees Celsius and 225rpm. 10 milliliters of media with the appropriate antibiotics was inoculated with 100 microliter of overnight culture and allowed to grown under the same environmental conditions until the culture density was 0.2~0.3 OD, approximately 3 hours. The 10 milliliter culture was split between 4 different sterile test-tubes, 2 milliliters of culture per tube. The conditions of test tubes A through D were as follows: A (-IPTG,-ncAA), B (-IPTG,+ncAA), C (+IPTG, -ncAA), and D (+IPTG, +ncAA). IPTG stock solution was made at 1000X concentration and the ncAA was added to yield 1 mM concentration. Sterile DI water was added in the place of ncAA and IPTG as a control. Once the controls, IPTG, and ncAA were added appropriately, the cultures were allowed to grow to 0.5 OD. 70 micro liters of each culture condition and control culture was added to a separate transparent wells for fluorescence and OD readings. |
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