Team:BGU Israel/Experiments
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<p><b>Note: We started the project with a different name for each approach. Eventually we came up with "P.A.S.E", but in our notebook we continued using the old ones for comfort sake: GK means P.A.S.E 1, and TB means P.A.S.E 2.</b></p></br></br> | <p><b>Note: We started the project with a different name for each approach. Eventually we came up with "P.A.S.E", but in our notebook we continued using the old ones for comfort sake: GK means P.A.S.E 1, and TB means P.A.S.E 2.</b></p></br></br> | ||
+ | <h7>28.7.2013 Analyzing GFP decline rate post induction</h7></br> | ||
+ | |||
+ | 1. A starter was diluted 1:50 with 10 ml LB, 10 µl CRB X 6 vials. | ||
+ | 2. The vials were put in incubation for 2 hours until the O.D. reached ~0.3 | ||
+ | 3. 1 mM of IPTG was inserted in each vial and the vials were returned to the incubator for 4 hours. | ||
+ | 4. Centrifugation for 10 min, 4000 RPM of the 6 vials. | ||
+ | 5. Separation of the fluid from the precipitation. | ||
+ | 6. Bacterial precipitation of each vial was mixed with 10 ml LB, 0.1 ml 50% Glucose and 10 µl CRB. | ||
+ | 7. Samples of the vials we taken to a 96 well plate and was put in the plate reader for 9 hours with 30 min intervals of O.D. and FLU examination. | ||
+ | |||
+ | Results:</br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/f/f7/Bgu_28.7.1.JPG" /></br></br> | ||
+ | |||
+ | An average of the 6 vials has been taken and the decline rate has been calculated by a graph of the decline period only. | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2013/d/dc/Bgu_28.7.2.JPG" /></br></br> | ||
+ | |||
+ | Conclusions:</br> | ||
+ | 1. Even though the medium with IPTG has been removed and replaced, the initial 2 hours showed a continued induction of IPTG on the expression of GFP. Possible explanation is that the IPTG that was in the bacteria diffused very slowly to the medium which didn’t contain any. Therefore, it is needed to take into account when calculating a specific duration of induction, the amount of time the inducer will diffuse out of the bacteria. | ||
+ | 2. After 2 hours there was a significant decline in FLU while the bacteria still in the Log phase. An equation of the decline rate was determined. The values of FLU/O.D. here declining because of the increase of O.D. while FLU stayed constant. This explains how GFP is diluted during bacterial growth and its FLU per cell declines by the equation listed in graph 2. | ||
+ | |||
<h7>29.7.2013 IPTG induction on GFP expression over time</h7></br> | <h7>29.7.2013 IPTG induction on GFP expression over time</h7></br> | ||
1. Starters were diluted by the following (amounts in ml):</br> | 1. Starters were diluted by the following (amounts in ml):</br> |
Revision as of 15:40, 26 October 2013
Experiments & Results
Read our well-documented notebook
Week 0 18.07.2013 - 24.07.2013
Week 1 25.07.2013 - 31.07.2013
Note: We started the project with a different name for each approach. Eventually we came up with "P.A.S.E", but in our notebook we continued using the old ones for comfort sake: GK means P.A.S.E 1, and TB means P.A.S.E 2.
- DNA volume - 1 uL.
- cuvette width - 0.2 cm.
- Time constant (Tao) - 5.5 msec.
- Two LB agar plates with cmp were plated with bacteria - one with 20 uL of culture, the other with 50uL. The plates were grown overnight in 300c, and on both colonies grew.
- 51 uL DDW.
- 15 uL KAPA HIFI buffer.
- 2.25 uL of dNTPs.
- 2.25 uL of forward primer.
- 2.25 uL of reverse primer.
- 1.5 uL of KAPA polymerase.
- The annealing temperature was set to 450C.
![](https://static.igem.org/mediawiki/2013/3/35/BGU_lab1.jpg)
Week 2 01.08.2013 - 10.08.2013
- Centrifuge 10 min at 4200 g and resuspend pellet in 300 μL ice cold 10% glycerol. (final Vol. 1:100)
- Add up to 100ng of the purified PCR product cassette (1 μL) to 40 μL of competent cells. 3 min on ice.
- Transfer the competent cells into an electroporation cuvette.
- Electroporate with Ec2 setting. #1 cuvette-> 5.8m/sec. #2 cuvette-> 5.9 m/sec.
- Plate 50 μL of culture on LB plates containing the appropriate antibiotic and/or selection agent. A serial of dilution was done, we use 104 – 108 to plate on the agar.
- Cuvette #1 was add to LB agar + IPTG 1mM, incubated at 37oC over night.
- Cuvette #2 was add to LB agar + IPTG 1mM+CMP 50mg/ml, incubate at 37oC over night, to confirm loss of pkd78.
- PkD 78
- pET 15b
- HiFi buffer X5
- DDW
- dNTP's
- ampR FWD & REV primer
- cmpR FWD & REV primer
- kappa HiFi polymerase
- DNA template
- Gel Agarose
![](https://static.igem.org/mediawiki/2013/d/da/BGU_ex42.png)
- Control sample was prepared – containing all components except the template.
- Each reaction was made twice.
![](https://static.igem.org/mediawiki/2013/7/7c/BGU_ex43.png)
![](https://static.igem.org/mediawiki/2013/8/84/BGU_ex44.png)
![](https://static.igem.org/mediawiki/2013/1/1d/BGU_ex45.png)
- Colony preparation – pick a colony and resuspend in 5 μl DDW PCR.
- Boil for 5 min at 99oC and immediately chill on ice (PCR program: colony_prep).
- Use the 5 μl as template for PCR.
![](https://static.igem.org/mediawiki/2013/3/33/BGU_ex46.png)
- Control sample was prepared – containing all components except the template.
- 10 samples were made – one from each colony.
![](https://static.igem.org/mediawiki/2013/0/0d/BGU_ex47.png)
![](https://static.igem.org/mediawiki/2013/f/fb/BGU_ex48.png)
- pkd78 – 151.1 μg ∼ 151.1 μl
- CRB gene – 169.8 μg ∼ 169.8 μl
- pkd78 – 455 μl
- CRB gene – 510 μl
- pkd78 – 151 μl
- CRB gene – 170 μl
- pkd78 backbone – 36 ng/μl
- CRB resistance gene – 19 ng/μl
![](https://static.igem.org/mediawiki/2013/8/89/BGU_ex49.png)
![](https://static.igem.org/mediawiki/2013/5/51/BGU_ex50.png)
![](https://static.igem.org/mediawiki/2013/4/42/BGU_ex51.png)
![](https://static.igem.org/mediawiki/2013/e/ed/BGU_ex52.png)
- The C1 plasmid arrived from hy-labs and was diluted. Final concentration - 50 ng\ul.
- Transformation of puc57-cI to super compotent cells (dh5α) by heat shock.
- Transformation of the pGFPuv into dh5α. This pGFPuv has undergone directed mutagenesis in order to add the stop codon.
- Transformation of C.O (copper oxidase) into BL21 that contain the UAA incorporation tRNA and the acRS (acetyl lysine synthethase). The machinery was on pSUP.
![](https://static.igem.org/mediawiki/2013/2/26/BGU_lab2.jpg)
Week 3 11.08.2013 - 17.08.2013
- 2 starters containing: 10 ml (LB), 10ul (CRB 100), 10ul (cmp 50), pkD78 + C1
- Kanamycin cassete has arrived.
- kan cassete PCR from pkD4 plasmid ( T annealing= 52oC).
- pkD78-ampR assembly - digestion of PCR products with XbaI and Xhol (FD)
- products were separated on agarose gel and extracted using gel extracting kit.
![](https://static.igem.org/mediawiki/2013/b/b2/BGU_ex36.png)
![](https://static.igem.org/mediawiki/2013/6/62/BGU_ex37.png)
- BL21 pkD78 + puc57-C1 +CRB100 (10ul) + CMP50 (10ul)
- BL21 puc57-C1 +CRB100 (10ul)
- PkD 78 PCR product
- CRB PCR product
- HiFi buffer X5
- DDW
- dNTP’s
- ampR FWD & REV primer
- cmpR FWD & REV primer
- kappa HiFi polymerase
- DNA template
- Gel Agarose
![](https://static.igem.org/mediawiki/2013/5/58/BGU_ex38.png)
![](https://static.igem.org/mediawiki/2013/9/9a/BGU_ex39.png)
- Sample 1 – BL21 pkD78 (EC) -> puc57-CI (1ul)
- Sample 2 – BL21(EC) -> puc57-Time Bomb (1.5ul)
- Sample 3 – BL21 (EC) -> puc57-CI (1ul)
- 2* CRB 100 CMP 50
- 5* CRB 100
- 3* KAN 30
- 35 uL DDW
- 10 uL HIFI buffer
- 1.5 uL rev+fwd primer (cI +his tag)
- dNTP 1.5 uL
- 1 uL DNA template (cI plasmid)
- 1uL KAPA polymerase.
![](https://static.igem.org/mediawiki/2013/d/d2/BGU_ex40.png)
![](https://static.igem.org/mediawiki/2013/a/a3/BGU_ex41.png)
![](https://static.igem.org/mediawiki/2013/7/70/BGU_lab4.jpg)
Week 4 18.08.2013 - 04.08.2013
- BL21 pkD78 + puc57-CI
- BL21+ puc57-Time Bomb
- BL21 + puc57-CI
![](https://static.igem.org/mediawiki/2013/b/bd/BGU_lab5.jpg)
Week 5 25.08.2013 - 31.08.2013
![](https://static.igem.org/mediawiki/2013/6/68/BGU_ex26.png)
![](https://static.igem.org/mediawiki/2013/4/4c/BGU_ex27.png)
![](https://static.igem.org/mediawiki/2013/0/0a/BGU_ex28.png)
![](https://static.igem.org/mediawiki/2013/2/22/BGU_ex29.png)
![](https://static.igem.org/mediawiki/2013/5/52/BGU_ex30.png)
![](https://static.igem.org/mediawiki/2013/7/72/BGU_ex31.png)
![](https://static.igem.org/mediawiki/2013/7/72/BGU_ex32.png)
![](https://static.igem.org/mediawiki/2013/5/5f/BGU_ex33.png)
![](https://static.igem.org/mediawiki/2013/4/44/BGU_ex34.png)
![](https://static.igem.org/mediawiki/2013/6/6a/BGU_ex35.png)
Week 6 01.09.2013 - 07.09.2013
![](https://static.igem.org/mediawiki/2013/2/24/BGU_ex25.png)
Week 7 08.09.2013 - 14.09.2013
![](https://static.igem.org/mediawiki/2013/2/26/BGU_ex19.png)
![](https://static.igem.org/mediawiki/2013/6/6f/BGU_ex20.png)
![](https://static.igem.org/mediawiki/2013/a/a6/BGU_ex21.png)
![](https://static.igem.org/mediawiki/2013/8/8f/BGU_ex22.png)
![](https://static.igem.org/mediawiki/2013/2/29/BGU_ex23.png)
- crbR 1 - 40 ng/μl
- crbR 2 - 10 ng/μl
- GK cassette - 17.5 ng/μl
![](https://static.igem.org/mediawiki/2013/0/0b/BGU_ex24.png)
- DPN1 treatment was performed on the PCR products from the 9\9\13. and electroporation was performed on the products and grown on 2 plates (50ul, 150ul)
- starter has been prepared from single colony - pkD78-amp + CRB100. - checking we have succeeded to replace the antibiotic resistance.
- one colony was taken from the plate made on the 10/9 in order to make a starter.
- 2 starters of BL21, pkD78, amp50, 30oC.
- 2 starters of BL21, pkD78 + puc57-cI, cmp50, amp100, 30oC.
- BL21 + pkD78
- BL21 + pkD78 + puc57-cI
Week 8 15.09.2013 - 21.09.2013
![](https://static.igem.org/mediawiki/2013/6/69/BGU_ex17.png)
![](https://static.igem.org/mediawiki/2013/6/69/BGU_ex18.png)
- 8 PCR tubes were prepared: 4 from puc57-cI sample, 4 from TB-cassette sample
- 1 PCR tube was used as blank.
- 10 μL of 5XKAPA HiFi Buffer
- 1.5 μL of dNTP mix
- 6.6 μL (125 ng) of Forward Primer + (125 ng) of Reverse Primer (primer is LAC1/araC , amplification PCR product at 8.9.13)
- 1 μL (5-50 ng) of DNA template (template is pGFPuv from 9.9.13)
- ddH2O to final concentration 50 μL
- Then add 1 μL of KAPA HiFi Polymerase.
- 10μl of competent cells + 2μl of mutagenesis product incubate on ice for 30min.
- Cells were transferred to water bath at 42C for 30sec and immediately back to ice for 2 min.
- 200 μl of SOC medium was added.
- Cells incubated at 37C for 1hr.
- Plated X μl on CRB plates over night at 37C. (50 μl plate X2, 100 μl plate X2)
![](https://static.igem.org/mediawiki/2013/3/33/BGU_ex1.png)
![](https://static.igem.org/mediawiki/2013/b/b4/BGU_ex2.png)
![](https://static.igem.org/mediawiki/2013/c/cf/BGU_ex3.png)
![](https://static.igem.org/mediawiki/2013/3/35/BGU_ex4.png)
![](https://static.igem.org/mediawiki/2013/1/1d/BGU_ex5.png)
![](https://static.igem.org/mediawiki/2013/5/58/BGU_ex6.png)
![](https://static.igem.org/mediawiki/2013/8/80/BGU_ex7.png)
![](https://static.igem.org/mediawiki/2013/c/ca/BGU_ex8.png)
![](https://static.igem.org/mediawiki/2013/e/ea/BGU_ex9.png)
![](https://static.igem.org/mediawiki/2013/a/ae/BGU_ex10.png)
![](https://static.igem.org/mediawiki/2013/f/fc/BGU_ex11.png)
![](https://static.igem.org/mediawiki/2013/1/1b/BGU_ex12.png)
![](https://static.igem.org/mediawiki/2013/f/f4/BGU_ex13.png)
![](https://static.igem.org/mediawiki/2013/3/34/BGU_ex14.png)
![](https://static.igem.org/mediawiki/2013/6/6d/BGU_ex15.png)
![](https://static.igem.org/mediawiki/2013/4/41/BGU_ex16.png)
- 10 μL of 5XKAPA HiFi Buffer
- 1.5 μL of dNTP mix
- 2.5 μL (250 ng) of Reverse+ Forward Primers (primer is LAC1/araC , done by Alex at 16.9.13)
- 1 μL (5-50 ng) of DNA template (template is pGFPuv from 9.9.13)
- 35 μL ddH2O to final concentration 50 μL
- Then add 1 μL of KAPA HiFi Polymerase.
- 1.5 μL Dpn1 was added to mutagenesis product
- Incubation at 37C for 1hr.
- 10μl of competent cells + 2μl of restriction product incubate on ice for 30min.
- Cells were transferred to water bath at 42C for 30sec and immediately back to ice for 2 min.
- 200 μl of SOC medium was added.
- Cells incubated at 37C for 1hr.
- Plated Xμl on CRB plates over night at 37C. (50μl plate X2, 100μl plate X2)
- digestion with restriction enzymes PstI and XbaI.
- Running restriction product in 2% agarose gel for B.bone extraction.
- QI Aquik gel extraction kit for purifying DNA from gel. (nanodrop: con. 16ng/ul)
- Ligation of the purifying B.Bone product with: cI T4, TB cassette, His tag+stop codon, KanR and, AmpR. (incubation for 1hr, 33C. stop reaction in 80C)
- Transformation by electroforetion to BL-21, incubation over night.
Week 9 22.09.2013 - 30.09.2013
- BioBricks: KanR, cI T4, HisTag+stop codon, TB cassette, and AmpR- PCR pruducts from 18.9.
- PSB1C3 Back Bone- back bone extraction product from 21.9.
- Restriction: using restriction enzymes PstI and XbaI to restricted each BioBrick for ligation with PSB1C3 Back Bone.
- NanoDrop measure of the restriction products.
- Ligation: using T4 ligase and T4 buffer for 16 hr, in room temperature, to maximized ligation products.
![](https://static.igem.org/mediawiki/2013/2/28/BGU_ex53.png)
![](https://static.igem.org/mediawiki/2013/2/26/BGU_ex54.png)
![](https://static.igem.org/mediawiki/2013/8/89/BGU_ex55.png)
![](https://static.igem.org/mediawiki/2013/6/65/BGU_ex56.png)
![](https://static.igem.org/mediawiki/2013/2/2c/BGU_ex57.png)
![](https://static.igem.org/mediawiki/2013/8/83/BGU_ex58.png)
![](https://static.igem.org/mediawiki/2013/e/e8/BGU_ex59.png)
![](https://static.igem.org/mediawiki/2013/a/a2/BGU_ex60.png)
![](https://static.igem.org/mediawiki/2013/3/30/BGU_ex61.png)
![](https://static.igem.org/mediawiki/2013/2/21/BGU_ex62.png)
![](https://static.igem.org/mediawiki/2013/7/7d/BGU_ex63.png)
- From the starter of BL21 pUC57 cI & pUC57 GK made yesterday (denoted a3), 1 ml was taken and diluted in 10 ml LB with crb, kan and 2mM IPTG. This culture was grown to OD 0.2 and plated on kan crb 2mM IPTG LB agar plates. The culture remaining was diluted – 0.1 ml of the culture was added to 9.9 ml LB with crb, kan and 2 Mm IPTG, and grown overnight.
- Bio Bricks transformation – Kan , cI TU, P.A.S.E 2 , cI +His With DH5α super compotent cells by heat shock. growin in 370C incubator.
- pkD78-crbR transformation with BL21 electro compotent using electroporation – growing in 300C incubator.
- Mini prep to DH5α – Kan , cI TU , P.A.S.E 2. yielding –
- Kan – 59.5 ng/&MU;l , 1.919 , 2.705
- cI TU – 44.5 ng/&MU;l , 1.935, 2.697
- P.A.S.E 2 – 75.5 ng/&MU;l, 1.936, 2.359
- Freezing DH5Α – Kan, cI TU, P.A.S.E 2. Using (-80) freezing protocol.
![](https://static.igem.org/mediawiki/2013/f/f1/BGU_ex64.png)
![](https://static.igem.org/mediawiki/2013/3/37/BGU_ex65.png)
![](https://static.igem.org/mediawiki/2013/7/7a/BGU_ex66.png)
![](https://static.igem.org/mediawiki/2013/b/b2/BGU_ex67.png)
![](https://static.igem.org/mediawiki/2013/c/cc/BGU_ex68.png)
![](https://static.igem.org/mediawiki/2013/1/18/BGU_ex69.png)
![](https://static.igem.org/mediawiki/2013/3/35/BGU_lab9.jpg)