Team meeting with PI and instructor, confirmed research topic on using butyrate to determine intestinal health
Team meeting with all student members, shared background knowledge from intestinal health and gene modification
Team meeting with PI and instructor, understood the methods of inserting the genes into the plasmid using enzyme cutting and linkage
Team meeting lab technicians and gastroenterologists, learned the basic protocols of lab safety and requirements for monitoring intestinal health
Team meeting with and instructor, developed the preliminary design of the device
Team meeting with device expert, determined the basic functions and operating environment of the device
Lab tour, learned to use lab equipment
Lab tour, learned to use lab equipment
- Prepared liquid (1% NaCl, 1% peptone, 0.5% yeast extract, desired volume of deionized water) and solid LB mediums (1% NaCl, 1% peptone, 0.5% yeast extract, 1.5% agarose, desired volume of deionized water)
- Sterilized the mediums with autoclave, added 1 ‰ benzylpenicillin Amp into the solid mediums
- Turned the mediums upside-down, cooled off the mediums then stored at 4℃
Enzyme cutting
- Added 10μL gene, 7μL sterile water, 2μL Buffer 2.1, 0.5μL EcoRI and 0.5μL SpeI
- Added 10μL gene, 7μL sterile water, 2μL Buffer 2.1, 0.5μL EcoRI and 0.5μL Xbal
- Sealed PCR tube, placed at 37℃ for 2h
Electrophoresis
- Added 25mL 1×TAE buffer solution with 0.25g agarose
- Heated for 2min, then added 2.5μL Genecolor
- Poured in gel cast and cooled off for 20min
- Placed gel in electrophoretic chamber and added 3μL of marker in first and last pore
- Added 1/5 of plasmid volume of 6×DNA Loading Buffer with plasmid, then injected into the pores of the gel
- Ran the gel under 110v for 35min
- Placed gel in gel imager for observation
Gel DNA extraction
- Cut the gel with DNA and placed in centrifugal tube, added 3 times the volume of gel of buffer DE-A
- Heated in water bath to melt the gel
- Transferred the solution into DNA preparation tube, centrifuged with 12000rpm for 1min and removed the waste liquid
- Added 500μL buffer W1, centrifuged with 12000rpm for 1min and removed the waste liquid
- Added 500μL buffer W1, centrifuged with 12000rpm for 30s and removed the waste liquid
- Added 500μL buffer W2, centrifuged with 12000rpm for 1min and removed the waste liquid
- Centrifuged with 12000rpm for 1min and removed the waste liquid
- Added 30μL of deionized water, placed in room temperature for 1min
- Centrifuged with 12000rpm for 1min, collected the plasmid in the centrifugal tube
Enzyme linkage
- Added 2μL 5×buffer, 0.4μL T4 DNA ligase, 2μL vector, 6μL target gene
- Placed in water in room temperature for 24h
- Added 50μL of sensory receptor cells with 5μL of plasmids, placed in ice for 20min allowing the DNA to adhere to the cell membrane
- Transformation using heat shock in 42℃ bath for 45s, placed in ice for 2min
- Added 500μL of liquid medium, placed in shaker at 37℃ for 1h
- Centrifuged with 7000rpm for 30s
- Removed 400μL of liquid above the bacteria and mixed the bacteria with the remaining liquid
- Added the bacteria onto the solid mediums, placed at 37℃ for 12h
Result: Partial success.
2 out of 6 solid mediums successfully grew bacterial colonies
- Picked the bacterial colonies from the solid mediums
- Injected the bacteria into the liquid mediums, placed in shaker at 37℃ for 12h
- Placed CP3 absorption column in collecting tube, added 500μL balance buffer BL, centrifuged with 12000rpm for 1min, then removed the buffer
- Added 2mL bacteria into centrifugal tube, centrifuged with 12000rpm for 1 min and removed the supernatant, repeat the process above
- Added 250μL P1 solution (with RNaseA) with bacteria sediment, mixed using pipette, then added P2 solution and flipped centrifugal tube gently for 8 times
- Added 350μL P3 solution and flipped centrifugal tube gently for 8 times to remove protein
- Centrifuged with 12000rpm for 10min, transferred supernatant into CP3 absorption column
- Centrifuged CP3 absorption column with 12000rpm for 1min, removed supernatant
- Added 600μL PW rinse (with pure alcohol), centrifuged with 12000rpm for 1min, removed supernatant
- Repeated the process above
- Centrifuged CP3 absorption column with 12000rpm for 2min, removed supernatant
- Placed CP3 absorption column into centrifugal tube, added 80μL deionized water, placed in room temperature for 2min
- Centrifuged with 12000rpm for 2min, collected the plasmid in the centrifugal tube
Repeated process of electrophoresis
Success. RFP was successfully inserted into vector (lanes 1, 2, 3, 5, 3400 bp approx.)
12 chambers for rapid assays, connected to middle chamber with tunnels for wastewater collection and disposal.
Assessment result: Approved
Assessment result: Unapproved (rotating optic components were unreliable, weak seal of device causes interference, no output components)
Repeated process of enzyme cutting, electrophoresis, gel DNA extraction, and enzyme linkup
Repeated process of plasmid cultivation on solid mediums
Repeated process of plasmid cultivation in liquid mediums
Repeated process of plasmid extraction
Repeated process of electrophoresis
Result: Fail. PLee promotor failed to insert into vector
Modification: increased the ratio of vector to pLee promotor from 1:3 to 1:5
Repeated process of enzyme cutting, electrophoresis, gel DNA extraction, and enzyme linkup with modification
Repeated process of plasmid cultivation on solid mediums
Repeated process of plasmid cultivation in liquid mediums
Repeated process of plasmid extraction
Repeated process of electrophoresis
Result: Success. pLee was successfully inserted into vector
Repeated process of enzyme cutting, electrophoresis, gel DNA extraction, and enzyme linkup with modification
Repeated process of plasmid cultivation on solid mediums
Repeated process of plasmid cultivation in liquid mediums
Repeated process of plasmid extraction
Repeated process of electrophoresis
Result: Fail. pPchA and PchA failed to insert into vector
Modification increased the ratio of vector to pPchA and PchA from 1:3 to 1:5
Repeated process of enzyme cutting, electrophoresis, gel DNA extraction, and enzyme linkup with modification
Repeated process of plasmid cultivation on solid mediums
Repeated process of plasmid cultivation in liquid mediums
Repeated process of plasmid extraction
Repeated process of electrophoresis
Result: Fail. pPchA and PchA failed to insert into vector
Modification: increased the ratio of vector to pPchA and PchA from 1:5 to 1:7
Repeated process of enzyme cutting, electrophoresis, gel DNA extraction, and enzyme linkup with modification
Repeated process of plasmid cultivation on solid mediums
Repeated process of plasmid cultivation in liquid mediums
Repeated process of plasmid extraction
Repeated process of electrophoresis
Result: Partial success. 1 out of 6 groups of pPchA and PchA was successfully inserted into vector
Modification: increased enzyme linkage time from 24h to 36h
Repeated process of enzyme cutting, electrophoresis, gel DNA extraction, and enzyme linkup with modification
Repeated process of plasmid cultivation on solid mediums
Repeated process of plasmid cultivation in liquid mediums
Repeated process of plasmid extraction
Repeated process of electrophoresis
Result: Success. pPchA and PchA was successfully inserted into vector
Team meeting with gastroenterologists and device experts, discussed improvements for device, including optic pathway design, temperature control, and chip replacement design
Modifications: fixed optic parts, changed chip replacement method from drawer to lid to ensure seal, added a screen for data output
Assessment result: Approved, authorized for production
Ran tests using sodium butyrate solutions with known concentration
Results: Success. There is a strong correlation between the displayed data from the device and the actual concentration of the solutions
