Team:PuiChing Macau/Notebook
Our Notebook
This is our notebook below, data collected throughout the whole year...
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Primer design and experiment preparation
1) Design and order primers for later on DNA extraction with products having the desired overhang/extra DNA
2) Culture medium preparation for later on use in transformation
1)Building and confirming construct
1)Amplify and extract fragments of K3503001 (alpha casein) K1608000 (SR Protein)
K1321014(cellulose binding domain), K3089023(mussel adhesion protein)
a) Confirmation with Gel electrophoresis
b) DNA purification (Gel extraction) of the correct DNA fragments from gel
2) Link pET with different overhangs : pET11a-Ptac-F, pET11a
a) Confirmation with Gel electrophoresis
3) The DNA fragments were then ligated to pET form three constructs Alpha casein(K3503001), CBD-Alpha casein(K3503008), mfp5-Alpha casein(K3503007), mfp5-SR(K3503006), CBD-SR(K3503004), ptac-SR-GST(K1608000)
a) DNA extraction with miniprep kit and confirm with PCR and gel electrophoresis then undergo transformation to E.coli.
4) Optimizing Restriction Endonuclease Reactions for (mfp5-SR), (mfp5-Alpha casein), (CBD- Alpha casein), (ptac-SR)
- Lane 1: Invitrogen trackit 1Kb plus DNA Ladder.
- Lane 2:pET-11a-mfp5-SR(K3503006)
- Lane 3:pET-11a-mfp5 alpha casein (K3503007)
- Lane 4: pET-11a-ptac GST-SR (K1608000)
- Lane 5:pET11a
1)Protein extraction (Alpha casein)
2)Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times
3)IPTG induction of Alpha casein(K3503001), mfp5-Alpha casein(k3503007), CBD-Alpha casein (K3503008)
(a)Incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed
4) Prepare for the SDS-Page Gel
5)Coomassie Blue stain
a) Bio rad protein dual color ladder, 2) pet11a, 3-4) SR(K1608000) 5-6) K3503006(mfp5-SR) 7-8) K3503004(CBD-SR). Lane 4,6,8 are with 1mM IPTG induction.)
6) Western blot
1)Protein extraction SR protein
2)Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times
3)IPTG induction of SR protein(K1608000), mfp5-SR protein(k3503006), CBD-SR protein (K3503004)
(a)Incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed.
4) Prepare for the SDS-Page Gel
5)Coomassie Blue stain
(Lane1) Bio-rad protein dual-color ladder, 2) pet11a, 3-4) K3503001(Alpha casein), 5-6) K3503007(mfp5-Alpha casein), 7-8) K3507008(CBD-Alpha casein). Lane 4,6,8 are with 0.1mM IPTG induction.)
6) Western blot
Check the fluorescent on the fabrics
1)We check the fluorescent on the fabrics with Nikon A1MP multiphoton confocal microscope and fluorescence confocal microscope.
2) We confirmed that the red fluorescence has an almost unobvious part
3)We finally choose RFP (red fluorescence protein) to add on our protein to check the adhesive.
1)Protein extraction (Alpha casein)
2)Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times
3)IPTG induction of Alpha casein(K3503001), mfp5-Alpha casein(k3503007), CBD-Alpha casein (K3503008)
(a)Incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed.
4) Prepare for the SDS-Page Gel
5)Coomassie Blue stain
(Lane 1)Bio rad protein dual color ladder, 2) pet11a, 3-4) SR(K1608000) 5-6) K3503006(mfp5-SR) 7-8) K3503004(CBD-SR). Lane 4,6,8 are with 1mM IPTG induction.)
Adhesion test
Comparing with bedsheet after washing and soaking
The percentage of the protein after processes
Fire testing
Vertical Burning Test using bedsheet
Fire retardant test for wood
IR spectrum between water and protein-coated bed sheets
IR spectrum of engineered flame retardant on bed sheet