Team:BGU Israel/Experiments
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+ | <h6 class="toggle-trigger"><a href="#"><b>Week 9</b> 22.09.2013 - 30.09.2013 </a></h6> | ||
+ | <div class="toggle-container"> | ||
+ | <div class="block"> | ||
+ | <div class="three-fourth "> | ||
+ | <h7>22.9.2013 Restriction and Ligation of BioBricks to PSB1C3 Back Bone</h7></br> | ||
+ | <ol class="bulletlist"> | ||
+ | <li class="bulletlist">BioBricks: KanR, cI T4, HisTag+stop codon, TB cassette, and AmpR- PCR pruducts from 18.9.</li> | ||
+ | <li class="bulletlist">PSB1C3 Back Bone- back bone extraction product from 21.9.</li> | ||
+ | <li class="bulletlist">Restriction: using restriction enzymes PstI and XbaI to restricted each BioBrick for ligation with PSB1C3 Back Bone. </li> | ||
+ | <li class="bulletlist">NanoDrop measure of the restriction products.</li> | ||
+ | <li class="bulletlist">Ligation: using T4 ligase and T4 buffer for 16 hr, in room temperature, to maximized ligation products.</li> | ||
+ | </ol> | ||
+ | </br></br> | ||
+ | |||
+ | <h7>24.9.2013 pKD78 back bone and crbR- PCR, restriction, ligation and transformation</h7></br> | ||
+ | <li class="bulletlist">pKD78 back bone amplification PCR, program "long fragment".</li> | ||
+ | <li class="bulletlist">analyzed products on 1.5% agarose gel and then purifying from gel using kit.</li> | ||
+ | </ol> | ||
+ | </br></br> | ||
+ | |||
+ | <h7>25.9.2013 pKD78 back bone and crbR- PCR, restriction, ligation and transformation</h7></br> | ||
+ | <li class="bulletlist">Restriction: using restriction enzymes XhoI and XbaI to restricted the purifying PCR products of pDK78 B.bone and crbR (from the 18.9).</li> | ||
+ | <li class="bulletlist">Both restriction products analyzed on 1.5% agarose gel and then purifying from gel using kit.</li> | ||
+ | <li class="bulletlist">Ligation (over nigth,room tem.) of the pKD78 back bone with crbR, using T4 ligase.</li> | ||
+ | |||
+ | </ol> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/2/28/BGU_ex53.png" /> | ||
+ | </br></br> | ||
+ | |||
+ | <h7>26.9.2013 PCR to make cI his tag gene a biobrick</h7></br> | ||
+ | pUC 57 cI his tag generated by site directed mutagenesis was used as a template to create a standard biobrick cassette to be inserted into psB1C3. The primers used are B his + stop FWD and B cI TU REV.</br></br> | ||
+ | Reaction mixture and cycle program:</br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/2/26/BGU_ex54.png" /> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/89/BGU_ex55.png" /></br></br> | ||
+ | The bands on the gel correspond to ~800 bp, which is about the size of cI his tag with biobrick standard prefix and suffix.</br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/6/65/BGU_ex56.png" /></br></br> | ||
+ | The purified cI his tag (using QIA quick PCR extraction kit)</br></br> | ||
+ | <b>Restriction and Ligation of BioBricks to PSB1C3 Back Bone</b></br></br> | ||
+ | 1. PCR products from 19.9 (kanR, cI TU, TB) and PCR product cI+his tag compatible (made by alex) were digested with Xbah and PST1 by the restriction protocol and by the following amounts:</br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/2/2c/BGU_ex57.png" /></br></br> | ||
+ | 2. After the restriction, the DNA was purified with the PCR purification kit. </br> | ||
+ | 3. pSB1C3 RFP plasmid was digested as well in the same protocol with the same restriction enzymes but using the green buffer.</br> | ||
+ | 4. After restriction, the DNA was loaded on agarose gel and the top bend was extracted with the QIAGEN extraction kit.</br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/83/BGU_ex58.png" /></br></br> | ||
+ | 5. Ligation of the inserts with the pSB1C3 backbone: </br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/e/e8/BGU_ex59.png" /> <img src="https://static.igem.org/mediawiki/2013/a/a2/BGU_ex60.png" /></br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/3/30/BGU_ex61.png" /> <img src="https://static.igem.org/mediawiki/2013/2/21/BGU_ex62.png" /></br></br> | ||
+ | |||
+ | <h7>26.9.2013 Final step of inserting the P.A.S.E. 1 system in BL21 cells</h7></br> | ||
+ | <b>1. Max induction with IPTG to produce maximum cI repressor</b></br> | ||
+ | 1.1 BL21 with pUC57 cI starter was grown over night.</br> | ||
+ | 1.2 The starter was diluted 1:50 into 3 vials with 10 ml LB and 10 µl CRB and incubated until it reached O.D. &tilde 0.2.</br> | ||
+ | 1.3 IPTG was added to each vial with different conc. ( 1mM, 2mM, 5mM).</br> | ||
+ | 1.4 The vials were put back in the incubator for 5 hrs of induction until they reach &tilde0.6 O.D.</br> | ||
+ | 1.5 Petri dishes were made with LB Agar that included 0.1% CRB, 0.05% Kan, 2mM IPTG.</br></br> | ||
+ | <b>2. Preparation of the cells for transformation</b></br> | ||
+ | 2.1 The vials were put in ice for 20 min and than in centrifugation for 15 min at 4000 RPM.</br> | ||
+ | 2.2 The supernatant was discarded and the cells were resuspended with 10 ml of 10% ice cold glycerol.</br> | ||
+ | 2.3 Centrifuge at 4000 RPM for 15 min and the supernatant was discarded.</br> | ||
+ | 2.4 The vials were resuspended with 5 ml of ice cold 10% glycerol and put back in centrifuge for 15 min at 4000 RPM.</br> | ||
+ | 2.5 The supernatant was discarded and the cells were resuspended with 400 µl of ice cold 10% glycerol and moved to 1.5 microfuge tubes.</br> | ||
+ | 2.6 Centrifuge at 10,000 RPM for 15 min in small centrifuge and discard of supernatant.</br> | ||
+ | 2.7 Resuspend with 90 µl of ice cold 10% glycerol.</br></br> | ||
+ | <b>3. Transformation of cells with GK cassette</b></br> | ||
+ | 3.1 Transformation was submitted by electroporation threw the Electotransformation protocol.</br> | ||
+ | 3.2 200 µl of 100mM IPTG were added to the plates made earlier (1.5).</br> | ||
+ | 3.3 150 µl of the cells were plated on plates and put in incubation for 12 hours.</br></br> | ||
+ | <b>4. Results </b></br> | ||
+ | Bacteria grew on all three plates. P.A.S.E 1 system insertion is complete!</br> | ||
+ | Starters were made from each plate with LB, kan50 and mM IPTG.</br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/7/7d/BGU_ex63.png" /></br></br> | ||
+ | |||
+ | <h7>27.9.2013 40 LB agar plates with kanamycin and 2mM IPTG were made.</h7></br> | ||
+ | Preparing BL21 pUC57 cI & pUC57 GK for self-destruct essay:</br> | ||
+ | <ol class="bulletlist"> | ||
+ | <li class="bulletlist">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.</li> | ||
+ | <li class="bulletlist">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.</li> | ||
+ | <li class="bulletlist">pkD78-crbR transformation with BL21 electro compotent using electroporation – growing in 300C incubator.</li> | ||
+ | <li class="bulletlist">Mini prep to DH5α – Kan , cI TU , P.A.S.E 2. | ||
+ | yielding – </li> | ||
+ | <ol class="bulletlist2"> | ||
+ | <li class="bulletlist2">Kan – 59.5 ng/&MU;l , 1.919 , 2.705</li> | ||
+ | <li class="bulletlist2">cI TU – 44.5 ng/&MU;l , 1.935, 2.697</li> | ||
+ | <li class="bulletlist2">P.A.S.E 2 – 75.5 ng/&MU;l, 1.936, 2.359</li> | ||
+ | </ol> | ||
+ | <li class="bulletlist">Freezing DH5Α – Kan, cI TU, P.A.S.E 2. Using (-80) freezing protocol.</li> | ||
+ | </ol> | ||
+ | </br></br> | ||
+ | |||
+ | <h7>28.9.2013 Colony PCR for cells that grew on plates after transformation with pUC57 GK</h7></br> | ||
+ | 1. Colony PCR was submitted in order to insure that the BL21 that grew on the plates after transforming GK cassette to BL21 with pUC57 cI worked and they contain the complete P.A.S.E 1 system.</br> | ||
+ | 2. One colony from the plate was taken and grew in 10 ml LB with 2mM IPTG and kan30 overnight.</br> | ||
+ | 3. The plasmids were extracted from the starter with the Invitrogen miniprep kit.</br> | ||
+ | 4. PCR for following templates:</br> | ||
+ | 4.1 plasmid extraction from the starter with GK primers</br> | ||
+ | 4.2 pUC57 GK with GK primers for control</br> | ||
+ | 4.3 plasmid extraction from the starter with cI TU primers</br> | ||
+ | 4.4 pUC57 cI with cI TU primers for control</br> | ||
+ | 5. Reaction Mixture and cycle program:</br> | ||
+ | 5.1 For GK presence (4.1 & 4.2):</br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/f/f1/BGU_ex64.png" /> <img src="https://static.igem.org/mediawiki/2013/3/37/BGU_ex65.png" /> | ||
+ | </br></br> | ||
+ | |||
+ | 5.2 For cI presence (4.3 & 4.4): </br></br> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/7/7a/BGU_ex66.png" /> <img src="https://static.igem.org/mediawiki/2013/b/b2/BGU_ex67.png" /> | ||
+ | </br></br> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="one-fourth last"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/3/35/BGU_lab1.jpg" /> | ||
</div> | </div> | ||
<div class="clear "></div> | <div class="clear "></div> | ||
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+ | </div> | ||
+ | |||
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Revision as of 16:08, 30 September 2013
Experiments & Results
Read our well-documented notebook
Week 1 29.07.2013 - 31.07.2013
- 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.
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
- Control sample was prepared – containing all components except the template.
- Each reaction was made twice.
- 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.
- Control sample was prepared – containing all components except the template.
- 10 samples were made – one from each colony.
- 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
- 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.
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.
- 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
- 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.
Week 4 18.08.2013 - 04.08.2013
- BL21 pkD78 + puc57-CI
- BL21+ puc57-Time Bomb
- BL21 + puc57-CI
Week 5 25.08.2013 - 31.08.2013
Week 6 01.09.2013 - 07.09.2013
Week 7 08.09.2013 - 14.09.2013
- crbR 1 - 40 ng/μl
- crbR 2 - 10 ng/μl
- GK cassette - 17.5 ng/μl
- 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
- 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)
- 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.
- 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.