Team:NCKU Tainan/Notebook Functional Test

Notebook Functional Test - iGEM NCKU

NOTE / Functional Test
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.