Team:Tianjin/Protocol

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Protocol

Plasmid Extraction

We use the TIANprep Mini Plasmid Kit made by TIANGEN Biotech Co.,Ltd. to extract plasmid. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume.
1. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).
2. Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
3. Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
Note: No cell clumps should be visible after resuspension ofthe pellet, otherwise incomplete lysis will lower yield and purity. 4. Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times.
Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
5. Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.
Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.
6. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
7. (Optional, actually we hardly ever use) Wash the Spin Column CP3 by adding 500 μl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and put Spin Column CP3 back to the collection tube.
This step is recommended to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
8. Wash the Spin Column CP3 by adding 600 μl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.
9. Repeat Step 8.
10. Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.
Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.
11. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.
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Week2(8/31/2016-9/6/2016)

  • We linked the remained cut CpxR-RFP fragment into the skeleton and then transformed the recombinant pUC57 and the pET21a into E.coli at the same time.
  • The transformation last night turned to be a failure. We tried it again.
  • The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.
  • We finally gave up the former design and decided to link the PETase gene into the plasmid pUC19. However, we did not have the key enzyme Sal1 so we started to construct the TPA positive feedback system.
  • We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the TPA-sensing leader sequence, PGK1 promoter, CYC1 terminator, RFP gene, TPA regulation protein gene (tpaR), TPA transporting protein gene (tpaK). Then we cut the fragments above and plasmid pRS413, pRS415, and pYES2 with corresponding enzymes and recycled the fragments from agarose gel.
  • We linked the fragments together by this way:
    1. pYES2-leader-PGK1-RFP.
    2. pRS413-PGK1-tpaK-CYC1.
    3. pRS415-PGK1-tpaR-CYC1
  • Then we used PCR to verify the success and all of the plasmids were correctly constructed. Then we transformed the there plasmids into Saccharomyces cerevisiae.
  • The key enzyme Sal1 arrived and we isolate the plasmid pET21a. Then we use BamH1 and Sal1 to cut both plasmid and PETase gene, then linked them together and transformed the recombinant plasmid into E.coli.
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    Week3(9/7/2016-9/13/2016)

  • We cultured the transformed E.coli and isolated the plasmid. Then we use PCR to amplify the whole fragment in pET21a from T7 promoter to T7 terminator. Then we recycled this fragment from agarose gel.
  • The transformed Saccharomyces cerevisiae had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.
  • We cut the T7 promoter-PETase gene-T7 terminator fragment with enzymes EcoR1 and Sac1. Then we linked it to the already cut plasmid pUC19 (cut in August 28th). Then we transformed the recombinant plasmid into E.coli.
  • We cultured the transformed Saccharomyces cerevisiae into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:
    1. Group 1: did not add TPA.
    2. Group 2: added 1000μL TPA standard solution.
    3. Group 3: added 100μL TPA standard solution.
    4. Group 4: added 10μL TPA standard solution.
    5.Group 5: added 1μL TPA standard solution.
  • We cultured the transformed E.coli into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of PETase gene.
  • We first detected the red fluorescence of E.coli, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.
  • The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.
  • We doubted that it might be the RFP in the kit was useless. We isolated the pET21a and used PCR to amplify the RFP gene.
  • We cut the RFP gene and pET21a with enzymes Xba1 and Sac1, then we linked them and transformed it into E.coli.
  • We cultured the transformed E.coli and added IPTG to induce the expression of RFP, and this time the red fluorescence was clear enough that could be seen directly.
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    Week4(9/14/2016-9/20/2016)

  • We started to construct another regulation way, the E.coli lysis regulation pathway. We first used colony PCR to obtain the ddpX gene from the E.coli genome and recycled the ddpX from the agarose gel.
  • We found that there was no enzyme cleavage site between the CpxR promoter and RFP gene in the part we use. We had to design the primers and amplified the CpxR promoter by PCR.
  • We used PCR to amplify the CpxR promoter. Then we recycled it from agarose gel.
  • We cut the CpxR promoter with enzymes Xba1 and BamH1, ddpX gene with enzymes BamH1 and EcoR1, first batch of pET21a with Xba1 and EcoR1, second batch of pET21a with BamH1 and EcoR1.
  • Then we linked these fragment in the following two ways:
    1. pET21a-CpxR-ddpX.
    2. pET21a-ddpX.
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    Week5(9/21/2016-9/27/2016)

  • We used PCR to amplify the whole fragments in pET21a (from CpxR to T7 terminator). However, the band in the agarose gel was disperse so that we were unable to recycle it.
  • We used colony PCR to verify if the pET21a had been correctly constructed, the result is yes.
  • We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.
  • We cultured the E.coli transformed into the pET21a-ddpX fragment and detect the OD600 in order to verify the lysis effection of ddpX.
  • Considering the pYES2 is multicopy plasmid so that the copy number would affect the RFP expression level, we decided to change the pYES2 to single-copy plasmid pRS416. Since the pRS416 does not have terminator in its backbone, we used PCR to amplify the CYC1 terminator from plasmid pYES2.
  • We cut the pYES2 with enzyme Hind3 and EcoR1, CYC1 with EcoR1 and Sal1, pRS416 with Hind3 and Sal1. Then we linked the three part together.
  • We transformed the three plasmids into Saccharomyces cerevisiae together.
  • The new primers using to amplify the CpxR-ddpX-T7 terminator fragment arrived and we redid the PCR. However, the disperse band was still existed.
  • The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate.
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    Week6(9/28/2016-10/2/2016)

  • We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with PETase gene and CpxR-RFP fragment showed the deepest red.
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