Team:KUAS Korea/Notebook
Notebook
General notebook
2018/12/26
1) set meeting order.
2) ideation
3) distributing roles
4) set monthly plan.
2018/1/3
Orientation Day!
we meet up with newcomers and introduced about our team members. Team leader ( Tae Hoon Lee ), Dry Lab leader (Jung Won Choi), Wet lab leader (Han HyunKyu), Human practice leader (Tehyun Eom)
2019/1/9
We met with professor In Geol Choi. Professor gaves some lecture about 'What is Synthetic biology' and 'DIY Bio'.
2019/1/16
We discussed about Igem ideas. Each have great Ideas such as Zinc finger Motif modification, fine dust remove tactics. Moss wall, and pet excreta smell treatment.
2019/1/23
We did Case study session about formal iGEM projects. Moreover, we discussed about our projects and give some feedbacks about them.
Also we set plan for the 2nd recruitment
- make poster
- contact with student center
- online poster upload to student information portal
- online poster upload to our Facebook and Instagram page
2019/1/30
Before the recruitment, we decided to open information session for volunteers. We choose date and contetns.>
Date
6 / 29
Contents
(1) Explain about synthetic biology
(2) Our new HP project ( Homecoming, collaboration)
(3) KUAS TV (youtube channel)
(4) What is iGEM?
2019/2/13
We redeemed our homepage. We made QR code and held poster on our homepage.
Also, we met professor and did Ideattion together.
2019/3/18
We met our new Recruit member.
2019/4/4
we checked our criteria for explanation and set the annual plan.
2019/6/30
Homecoming DAY!
we met some former igemers and we held some sessions to talk about synthetic biology. Also We can share informations about synthetic biology's future.
2019/7/19 ~ 2019/7/24
DH10B competent cell preparation
2019/7/25
competent cell TE test(transformation)
To calculate transformation efficiency, we transformated pB3 vector DNA. Then we give heat shock during 1 min and 30 min recovery. After that, we spread these DNA to LB ampicillin plate.
2019/7/26
Competent cell TE measurement
We noticed that there are many colonies on the plate. (The value of TE is 5.23E+3, DH10B+pB3)
colony select & culture
We try to get vector from plate, we select two colony randomly, and we inoculate these to 15ml LB broth(liquid). Then conduct shaking incubation. After 5 hours, we frozen at 4℃
2019/7/29
minipreparation
We did plasmid extraction with cell culture. The density of sample one was 59.560ng/ul, sample 2 was 61.455ng/ul
electrophoresis
Only sample 2 shows valid result. ( We checked band on the sample 2.)
2019/7/30
Transformation
To measure TE value over time, we transformed DH10B with sample 2 pB3 DNA and spread again. We changed heat shock time each 0s/10s/30s/1min.
2019/7/31
colony count
We checked colonies for all plate. (0s – 434, 10s- 516, 30s – 324, 1min – 283)
colony select & culture
To extract pB3 vector, we choose 2 colony randomly from 60sec plate. Then we Inoculate them to 10ml LB broth(liquid) and conducted shacking incubation for 5 hours. Then we frozen them in 4℃.
2019/8/01
minipreparation
We extracted plasmid from Cultures grown on 31st. Sample 1 was 46.343ng/ul, sample 2 was 46.512ng/ul
electrophoresis
We checked valid band on the both sample successfully.
2019/8/6
FGI(focused group interview) with STEPI
We met professor and researcher
2019/8/7
transformation
To get PB vector series (pB1, pB7), transformed each plasmid DNA(5ul) to competent cell and Spread to LB-amp.
2019/8/8
colony select and culture
Cell was grown in the pB6, pB7. So we choose one colony then inoculation to 15ml LB broth(liquid). Then we conducted shacking incubation with 5 hours and put them into refrigerator
2019/8/9
minipreparation
we extracted plasmid from cell broth, which was grown during 24hours. p86 was 90.742ng/㎕ and pB7 was 70.682ng/㎕
electrophoresis
We checked valid plasmid band from both samples.
transformation
To get plasmid again, we transformed again. Then we changde recovery time 30min to 1hr.
2019.8.10
colony check
we checked colonies from all plate
2019/8/12
colony selection, culture and minipreparation
We choose one colony from plate which was checked at 8/10. Then we inoculate them in to 10ml LB broth(liquid) and conducted shacking incubation during 5 hours.
plasmid extraction
Then we extracted plasmid from culture. pB1: 27.087ng/㎕, pB2: 27.201ng/㎕, pB3: 21.234ng/㎕, pB4: 23.218ng/㎕, pB5: 21.298ng/㎕, pB6: 19.266ng/㎕, pB7: 21.398ng/㎕
enzyme cut & electrophoresis
To ensure that sma I site is existing in the pB vector’s cloning site, we cut Plasmid with sma I. Then conducted electrophoresis. We found single band for all plasmids. But, pB4’s band was very faint
2019/8/14
DNA(gBlock)primer resuspension
To calibrate gBlock(luc2, hydroxy)’s concentration, we resuspended each gBlock with 200ul, and 400ul. Also we resuspended primers to become 100pM. luc2_pB_R(277㎕), hydroxy_pB_F(346㎕), hydroxy_pB_R(262㎕). After that, we move 5ul for each solution to tubes and diluted with 45ul EB.(10x dilution, total volume 50ul)
PCR
Mix gBlock(luc2, hydroxy), primer, Q5 DNA polyemerase, Q5 reaction buffer. Then run the PCR. By electrophoresis, we checked valid band was exposed. (luc2, 747bp, hydroxy.1281bp)
Vector enzyme cutting
Use sma I to pB series(pB3~pB7) as restriction enzyme and checked 1 band through electrophoresis.
clean up the products
Clean up the pB vector series(pB3~pB7) and check the concentration. ; luc2: 99.371ng/㎕, hydroxy: 60.118ng/㎕, pB3: 16.051ng/㎕, pB4: 5.037ng/㎕, pB5: 7.078ng/㎕, pB6: 32.304ng/㎕, pB7: 11.156ng/㎕
DNA assembly
WE used T4 DNA polymerase LIC(ligation independent cloning) method for recombination. Molar ration of Luc2 was 1:5, and hydroxy was 1:3. Then we put enzyme for 2.5 min in room temperature. Then iced to each case including 5 negative control.
transformation
After assembly, we transformed DNAs to DH10B competent cell. Then, spread these cell to LB-amp plate.(heat shock 30sec, recovery 1hr)
2019/8/15
we checked colony in Luc2-pB7, hydroxy pB3
2019/8/20
Colony PCR
We conducted colony PCR by pfu DNA polymerase. The result was failed
2019/8/21
colony PCR
We conducted colony PCR again, then we re-checked result by electrophoresis. Luc2 was 3 of 3, and hydroxy was 7 of 7
colony culture
We used 1ml culture as starter which was used in colony PCR. Then we scaled up 10ml and conducted shaking incubation.
2019/8/22
minipreparation
We used 21th culture for plasmid extract. We extracted two plasmids per gBlock. luc2-pB7_1: 32.304ng/㎕, luc2-pB7_2: 31.440ng/㎕, hydroxy-pB3_1: 28.090ng/㎕, hydroxy-pB3_2; 33.246ng/㎕
transformation
We get 4 different vector and transformed them in to BL21(DE3). Then spread them into LB-amp
2019/8/23
transformation
The result of 22nd transformation was not clear. So we conducted transformation again. This time, we extended recovery time over 1hr.
2019/8/24
we checked colony for all plates.
2019/8/26
overexpression
we choose one colony for each 7 plates.Then cultured 10ml for each. Then treated with IPTG(without OD measure) Then conducted incubation at 16℃
2019/8/27
Sonication
Centrifugate overexpressed culture(4℃, 2000g, 20min) and sonicate(80W, 20sec sonicating/40sec restin, repeat 3 times) the culture after resuspend with 600㎕ lysis buffer. After it, keep it in two groups, pellet and supernatant, in 4℃
2019/8/28
Solubility test faliure
2019/8/29
Miniprep
Extract plasmid from the colony which newly cloned 28th, checked with colony PCR and cultured after scaled up
2019/9/3
transformation
; Transformate the 6 plasmids(luc2-pB3_4, luc2-pB4_2, hydroxy-pB3_3, hydroxy-pB3_4, hydroxy-pB4_3, hydroxy-pB4_6) which extracted at 29th clone and chosen arbitrarily, to Bl21(de3)-star(expression strain). Then spread on LB-AMP agar plate.
SDS_PAGE gel preparation
for expression test (reference: protein method second edition)
2019/9/4
Expression test(by assistant)
pick 3 colonies per each plasmids(Luc2-pB3, Suc2-pB4, hydroxyl-pB3, hydroxyl-pB4), and conduct IPTG induction(1mM) at 37℃, 3hr. Then heat it at 90℃ using PCR machine to lyse cell, conduct SDS-PAGE;control group-culture which didn’t conduct IPTG induction. As a result, there was a significant difference with control in the band of the desired size.
SDS-PAGE
for solubility test
2019/9/5
Pick one from each samples of 9/4 and over express them.
2019/9/6
solubility test
Sonication
Centrifuge and remove the supernatant of cultures, mix the pellets with SDS-PAGE buffer. Conduct 1sec sonicating-3sec resting at 80W, repeat it 30 times.
Centrifugation
Centrifuge except the total solution. Devide supernatant and pellet and resuspense pellet with buffer.
SDS-PAGE
Run samples and ladders(total 44). As a result, proteins that expressed from the DNA assembled at pB4 appeared at both Luc2 and hydroxy in soluble part.
2019/9/9
Over express Luc2-pB4, htdroxy-pB4 at 16℃(1mM IPTG), to purificate by the result of 9/7
2019/9/10
Centrifugate 9/9 culture and only keep pellet frozen
2019/9/16
Prepare LB-AMP plate for new gBlock(nnLuz, cph) cloning.
2019/9/17
Prepare LB-CHL plate for characterization
2019/9/18
Transformation using RFP coding device parts.
2019/9/19
Cannot identify any colony as a result of transformation at 9/18.
2019/9/20
Protein purification failure
retry from the transformation.
Retry the transformation for characterization. Increase the amount of DNA.
2019/9/21
As a result of transformation at 9/20, there’re only one white colony grow up, on 24P.
2019/9/22
Conduct additional transformation for characterization.As 24P colony appeared red, picked it and culture for 7 hours, conduct miniprep
2019/9/23
transformation: conduct transformation. Luc2-pB4 to BL21(DE3)-star and hydroxyl-pB4 to C43(DE3)
2019/9/24
The experiment that conducted on 9/23 all succeed. Picked each colony and made starter.
2019/9/25
Cloning
by assistant. Cloned new gBlock nnLuz, cph using T4 DNAP LIC method.
2019/9/26
minipreparation
secure nnLuz, cph DNA, cloning at 9/25
2019/9/27
Transformation
retransform the 23O, 24P DNA which conducted miniprep to DH10B
2019/9/28
Both 23O, 24P which transformed on 9/23 appeared red. Picked each colony and incubate until saturation. Then keep it cold.
Add the starter which made on 9/24 to TB 200ml and set OD600=0.8. Keep it in shaking incubator for 3 day.
2019/10/1
saturation density measurement&transformation: Add 23O, 24P (cultured on 9/28) to LB and scaleup. Culture it enough and measure the OD600. Dilute the original culture medium and spread it on LB-CHL plate
2019/10/2
TE
colony counting
OD600/cell number relation
calculate the result by colony counting; About 1687 colony appear when spread the 40㎕ culture that dilute E-4 the culture 23O-DH10B, which OD600=0.418 at 1/5 dilution. It means, there were about 4.2175*E8 viable cells exist in original culture. In the same vein, there were about 5.4375*E8 viable cells exist in 24P of OD600=0.596.
2019/10/4
Centrifugation
centrifuge overexpressed culture (at 9/28) 3500g, 4℃, 20min and remove supernatant, keep it at -20℃
2019/10/7
Transformation
nnLuz-pB4/cph-pB4*BL21(DE3)-star/C43(DE3) for expression test, and 23O, 24P transformation for fluorescence measurement
Standard curve
measure by dilution. mRFP1 LPETG protein(0.51mg/ml), RLU E-2~E-11.
2019/10/8
Successed all transformation conducted on 10/7. Pick colony for expression and culture it in LB broth. Then keep only pellet(4000g, 10min) at -20℃.
2019/10/10
Culture C43(DE3) for one day for activity test
Characterization: Culture 23O, 24P single colonies in 10/7 plate for 6 hours. Then measure the OD600 of diluted(1/5) suspension. Set the suspensions to OD600=1 and load 100㎕ to 96well plate, measure RLU 530nm/590nm
2019/10/11
Centrifuge C43(DE3) culture on 10/10. Then conduct resuspension it with 4L2/4H6+C43(DE3) pellet cultured on 9/28 and sonicate the suspension. Centrifuge this suspension and 4L2/4H6+BL21(DE3)-star suspension made on 9/20 and take only supernatant. Mix crude extract+NADPH+hispidin and measure luminescent using luminometer.