Notebook - Functional Test
Compare promoter efficiency of Pcar with PI
May 14/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-Pcar-RBS-amilCP-TT and pMD19T-PI-RBS-amilCP-TT from LB agar plate .
May 15/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
May 16/2016
Induction : add 100 μl of bacteria culture to 96 well plate and add glucose solution to final concentration : 0, 5, 15, 30, 60, 120 mM
Readouts: measure OD 580 kinetically for 3.5 hr and compare the efficiency of Pcar and PI
Glucose-Color detection test
May 20/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PI-RBS-amilCP-TT from LB agar plate .
May 21/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
Induction : add 100 μl of bacteria culture to 96 well plate and add glucose solution to final concentration : 0, 5, 15, 30, 60, 120 mM.
Readouts: measure OD 580 kinetically for 3.5 hr.
Urine Glucose-Color detection test
Jun. 17/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PI-RBS-amilCP-TT from LB agar plate .
Jun. 18/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
Sample preparation :collect urine sample from non-diabetic healthy individuals , and add additional glucose to final urine glucose concentration of 0,10,30,120,240 mM.
Induction : add 100 μl of bacteria culture to 96 well plate and add Sample Urine to final concentration : 0, 5, 15, 30, 60, 120 mM.
Readouts: measure OD 580 kinetically for 3.5 hr.
2-hour Glucose-Fluoresence detection test
Jul. 15/2016
Transformed plasmid (pMD19T-PpI-B0034-E1010) to BL21 competent cell.
Jul. 16/2016
Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one colony from the agar plate.
Jul. 17/2016
Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-PI-RBS-mRFP was correct.
Culture: 10 ml of LB (with Ampicillin) cultured with 100 μl pre-cultured bacteria.
Jul. 18/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PI-RBS-mRFP from LB agar plate .
Jul. 19/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
Induction : add 100 μl of bacteria culture to 96 well plate and add glucose solution to final concentration : 0, 5, 15, 30, 60, 120 mM.
Readouts: measure fluorescence of excitation 530 / emission 590 kinetically for 2 hr.
12-hour Glucose-Fluoresence detection test
Jul. 25/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PI-RBS-mRFP-TT from LB agar plate .
Jul. 26/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
Induction : add 100 μl of bacteria culture to 96 well plate and add glucose solution to final concentration : 0, 5, 15, 30, 60, 120 mM.
Readouts: measure fluorescence of excitation 530 / emission 590 kinetically for 12 hr.
Lysis test
Aug. 22/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PBAD-RBS-lysis-TT from LB agar plate .
Aug. 23/2016
Transfer : 20 ml of modified M9 (with Ampicilin) added with different glucose/arabinose concentration and culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 15 mins and transfer to 96 well plate.
Readouts: measure OD 600 kinetically with plate readerfor 4 hours .
Lysis test-2
Sep. 01/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PBAD-RBS-lysis-TT from LB agar plate .
Sep. 02/2016
Transfer : 20 ml of modified M9 (with Ampicilin)added with different glucose/arabinose concentration and culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 15 mins and transfer to 96 well plate.
Readouts: measure OD 600 kinetically with plate readerfor 4 hours .
2-hour Urine Glucose-Fluoresence detection test
Sep. 05/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PI-RBS-mRFP-TT from LB agar plate .
Sep. 06/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
Sample preparation :collect urine sample from non-diabetic healthy individuals , and add additional glucose to final urine glucose concentration of 0,10,30,120,240 mM.
Induction : add 100 ul of bacteria culture to 96 well plate and add urine sample to final concentration : 0, 5, 15, 30, 60, 120 mM
Readouts: measure fluorescence of excitation 530 / emission 590 kinetically for 2 hr.
12-hour Glucose-Fluoresence detection test-1
Sep. 12/2016
Pre-culture: 5 ml of LB (with Ampicilin) was inoculated with one colony of pMD19T-PI-RBS-mRFP-TT from LB agar plate .
Sep. 13/2016
Culture: 20 ml of modified M9 (with Ampicilin) culture with 200μl pre-cultured bacteria. Grow up at 37 degree for 4 hours and test OD 600 until it reached 0.55~0.65.
Sample preparation :collect urine sample from non-diabetic healthy individuals , and add additional glucose to final urine glucose concentration of 0,10,30,120,240 mM.
Induction : add 100 μl of bacteria culture to 96 well plate and add urine sample to final concentration : 0, 5, 15, 30, 60, 120 mM.
Readouts: measure fluorescence of excitation 530 / emission 590 kinetically for 12 hr.
