Team:PuiChing Macau/Notebook
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Order Plasmid and Experiment Preparation
BBa_K3863004 Phac-flag-PCT(optimized)-HA-his
BBa_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his
GFP (J364000)
1.Exfection ™ EF Midi:
2.RT-qPCR:
- a.BBa_K3863007 PHA-flag-PCT-HA
- b.BBa_K3863004 Phac-flag-PCT(optimized)-HA-his
- c.BBa_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his
- d.BBa_K3863003 fabB-his
- e.BBa_K3863006 a-amylase-HA-glucoamylase-his
BBa_K3863006: a-amylase-HA-glucoamylase-his
- Transformation of E.coli
- Pick Colony & Bacteria Culture Preparation
Testing amounts of monosaccharides:
Glucose test result to compare the change of the amount of glucose between day 1 and day 2 in the addition of amylase and glucoamylase
E.coli growth analysis in food waste:
BBa_J3640000: Green Fluorescence Protein (GFP) BL21 (control)
1.Establishing model of E.coli growth in food waste with GFP and O.D.600 (with starch and without starch)
BBa_K3863003 fabB-his
BL21 (control)
Investigating Acid-Tolerant System (through CFU colony-forming unit):
- 1.Transformation of E. coli
- 2.Pick colony
- 3.IPTG Induction and Bacterial Culture Preparation
- a. BBa_K3863003 fabB-his
- i. uninduced
- ii. 0.1mM IPTG
- b. BL21
- i. uninduced
- ii. 0.1mM IPTG
- 4.Spread plate and Count Colony
- Investigating acid tolerant system, to see if the addition of fabB is capable for E.coli’s growth in acidic environment through colony forming unit (C.F.U).
BBa_K3863004 Phac-flag-PCT(optimized)-HA-his
BBa_K3863007 PHA-flag-PCT-HA
pETDuet (vector control)
Bioplastic production (without phasin):
- 1. BBa_K3863004 Phac-flag-PCT(optimized)-HA-his (BL21)
- a. UN: uninduced
- b. IN: 0.1mM IPTG
- 2. BBa_K3863007 PHA-flag-PCT-HA (BL21)
- a. UN: uninduced
- b. IN: 0.1mM IPTG
- 3. pETDuet (vector control)
- a. UN: uninduced
- b. IN: 0.1mM IPTG
Verifying Bioplastic Production through IR(Infrared Radiation) Spectroscopy:
To test if PHA(K3863007), Phac(BBa_K3863004) works for bioplastic production.
BBa_K3863004 Phac-flag-PCT(optimized)-HA-his
BBa_K3863007 PHA-flag-PCT-HA
BBa_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his
pETDuet (vector control)
Bioplastic production (with phasin):
- Compare the result with and without phasin, to see if the yield of bioplastic production has increased with the addition of phasin.
- 1. Produce Bioplastic Culture
- 2. Producing Bioplastic
- 3. Bioplastic Extraction
- 4. BBa_K3863004 Phac-flag-PCT(optimized)-HA-his (BL21)
- a. UN: uninduced
- b. IN: 0.1mM IPTG
- 5. BBa_K3863007 PHA-flag-PCT-HA (BL21)
- a. UN: uninduced
- b. IN: 0.1mM IPTG
- 6. BBa_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his
- a. UN: uninduced
- b. IN: 0.1mM IPTG
- 7. pETDuet (vector control)
- a. UN: uninduced
- b. IN: 0.1mM IPTG
Sample:
Nile Red - quantitative measurement of bioplastic production:
- 1.BBa_K3863004 Phac-flag-PCT(optimized)-HA-his
- 2.BBa_K3863007 PHA-flag-PCT-HA
- 3.BBa_K3863008 Phac-flag-PCT(optimized)-HA-phasin-his
- 4.pETDuet (vector control)
- Compare the result with and without phasin, to see if the yield of bioplastic production has increased with the addition of phasin through quantitative measurement by measuring the intensity of Nile Red.