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Team:SYSU-China/page project notes
From 2011.igem.org
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Experiments Dairy
Inoculate 10mL LB with the E. Coli BL21 plys strain.
Preserve the E. Coli BL21 plys strain in 4℃ incubator.
1. Temperature gradient PCR of the two genes, trkD and cheZ, and two promoters, recA and recN promoters, according to their primers' Tm.
2. Result: The best PCR annealing temperature of trkD and cheZ are 60°and 45°respectively. The PCR of recA and recN promoters will be repeated tomorrow.
1. PCR of trkD and cheZ to get large amount of these genes, but failed because of mistake of wrong PCR system.
2. PCR of recA and recN promoters, the best PCR temperature are both 60°.
3. Repeat the PCR of trkD and cheZ and extract pUC18 plasmid. Success!
Cut the protective bases of the restriction sites of EcoRI and XbaI, and tried to link the promoters to the genes but failed.
Cut the plasmid with the enzyme of EcoRI, and link the promoters and genes and the plasmid together over night in 16°.
Eletrotransform the linked product to DH5a competence cells and revive with LB and culture them on the LB plate. However, there are no growing cells on the plate, which means our first try failed.
PCR of the two promoters and the two genes, but during the gel extraction process, a small accident happened and the covers of the Ep tubes became powder, which blocked us from distinguish our four products. But we conducted a electrophoresis of our four products and we can differ our products.
Cut the protective bases of the restriction sites of EcoRI and XbaI, and cut the plasmid with the enzyme of EcoRI, and link the promoters and genes and the plasmid together over night in 16°.
Eletrotransform the linked product to DH5a competence cells and revive with LB and culture them on the LB plate. However, there are no growing cells on the plate, which means our first try failed.
1. Use the PCR products two days ago and cut the restriction sites of them and linearize the plasmid, and link the promoters, the genes and the plasmid in 16° overnight.
2. Re-PCR recN since the first design of it has some mistake.
Successfully link recA promoter to the gene of cheZ (there are colonies on the plate and positive results of Conlony PCR). Then we sent the strain to Invitrogen to check its sequence if there is any base mutation. It turned out that the promoter and the gene are normal. Till now, we get our first useful plasmid: recA-cheZ-pUC18.
We tried to construct the plasmid with GFP. We design the restriction sites of the GFP to be PstI and HindIII, and then we purified the GFP and linked it to the linearized pUC18.
1. Eletrotransform the linking product to DH5a competence cells, and grow them on plate.
2. We continued to link recN to trkD. We PCR the two parts and cut them with EcoRI and XbaI, and cut the pUC18 plasmid with EcoRI. And links them at 16°overnight.
1. There is no colony on the plate.
2. Eletrotransform the linking product of recN and trkD to DH5a competence cells, and grow them on plate.
We sent the two strains to check sequence.
The recN and trkD did not link into the plasmid by checking the sequencing result.
1. We ran a temperature gradient PCR of the new cheZ and trkD, both of which are without terminator because we want to link the two genes before the GFP and exam the gene expression by observe the fluorescence of GFP. However, the PCR of trkD(no terminator) failed while the cheZ(no termination ) succeed.
2. Cut the plasmid of pUC18 with HindIII and PstI, then exam the product with the GFP gene. Then link the gene of GFP with the plasmid of pUC18.
Eletrotransform the linking product into DH5a cells and culture them on an Amp plate. Patch again after 12h culture.
1. Colony PCR of the colonies on the plate of GFP-pUC18 and get 3 positive results. Inoculate 3 colonies into 5mL LB and place them in the shaker of 220rpm 37°overnight.
2. PCR of CheZ and trkD, both of which are off terminators, and the two promoters. Then use enzymes to cut the protective sites of the genes and the restriction sites of the plasmid of GFP-pUC18. Then link recA, recN, cheZ, trkD respectively with the plasmid.
1. Double digest pUC18 and EGFP with HindIII and PstI, then ligate pUC18 and EGFP with T4 ligase in 16℃ over night. (1 is 1Kb marker, 2 is pUC18 and 3 is EGFP).
2. Gradient PCR to search the proper condition of cloning trkD and cheZ (for western blot). (Left side is cheZ, right side is trkD).
1. Send the 3 GFP-pUC18 strains to Invitrogen to check their sequence. And we extract plasmid from the leaved inocula.
2. Eletrotransform the linking product into DH5a cells and culture them on Amp plates. Patch again after 12h culture.
1. Transformation of pUC18-EGFP to DH5α.
2. Use KOD plus polymerase to amplifying trkD and cheZ (for western blot).
3. Double digest trkD and cheZ (for western blot) with EcoRI and HindIII, then ligate them to pET-28a and pET-32a which were digested with same enzymes. However, with few units of plasmids, this ligation had to be done in next day.
1. Re-digest pET-28a and pET-32a with EcoRI and HindIII, then ligate to trkD and cheZ (for western blot) with T4 ligase in 16℃ over night. (1 is cheZ, 2 is trkD, 3 is 1Kb marker, 4 is pET28a, 5 is pET-32a).
2. PCR trkD (for recombinant PCR), but failed.
3. Clony PCR pUC18-EGFP with Go Taq® to test the result of transformation.
Patch the colonies on the plates and run a colony PCR and got several positive result .Inoculate the positive strain with 5mL LB respectively.
Send our samples to check their sequence. Then extract plasmids from those different strains.
Cut the different plasmids and exam whether these plasmids have genes in them. The cheZ-GFP-pUC18 and trkD-GFP-pUC18 do have the two genes on the plasmid, while the two promoters failed.
Try to use recombinant PCR to clone recN-trkD in one-step program and two-step program with Go Taq®, which helps to find the proper condition of PCR.
1. Digest plasimds cheZ-pET-32a and trkd-pET-32a with EcoRI and double digest with EcoRI and HindIII to test whether their lengths are correct, and the result shows trkd-pET-32a is correctly, but cheZ-pET-32a is not.
2. Try to use recombinant PCR to clone recN-trkD in two-step program with Go Taq®, which helps to find the proper condition of PCR.
Induce cheZ-pET-32a and trkd-pET-32a with 0.1, 0.5, 1.0 mM IPTG in OD 0.600.
In SDS-PAGE, use proteins in liquid and lysate expressed by cheZ-pET-32a and trkd-pET-32a to do electrophoresis. And the gel with proteins in liquid does western blot, while the other gel is dyed with G250. (1~4 are trkd-pET-32a induced by 0, 0.1, 0.5, 1.0 mM IPTG, 5 is marker, 6~9 are cheZ-pET-32a induced by 0, 0.1, 0.5, 1.0 mM IPTG).
We inoculated the cheZ-GFP-pUC18 and trkD-GFP-pUC18 with 5mL LB. We use IPTG to trigger the lac promoter before both the cheZ-GFP-pUC18 and trkD-GFP-pUC18, then use fluorescence microscope to exam whether the two genes have successfully expressed.
We intermediatecultured 1ml strains with 100mL LB. After two hours shaking and measured the OD value of the two strains of 0.45, we separated the 100ml inocula into 4 conical flasks and induced with 3 different concentrations of IPTG(one leaved is control). After 4 hours induction, we used fluorescence microscope to exam the inocula and find out that the control group, which should not be of a dark view, have fluorescence too. That means the lac promoter can trigger the expression of its downstream genes without induction. Then we want to transfer our genes onto another plasmid, pET28a, whose promoter lied between the restriction sites of BamHI and BglII. The two enzymes are isocaudamers and the gap cut by them can be linked without inserting any sequence.
Clone recN-trkD recombination in recombinant PCR program with PrimeStar®. After double digest the gene with EcoRI and PstI, ligate it with plasmid pUC18-EGFP digested with same enzymes. (1 is 1Kb marker, 2 is recN-trkD, 3 is pUC18-EGFP).
1. Cut the plasmid of GFP-pUC18 and the protective sites of recN and recA with the enzyme of XbaI and EcoRI, but the plasmid is so little amount that it cannot be seen on the gel. Inoculate the strain with the plasmid of GFP-pUC18.
2. Use BamHI and BglII to cut the plasmid of pET28a, and use T4 ligation enzyme to link the gap cut by the two enzymes of the plasmid.
1. Eletrotransform the linking product into DH5a cells and culture them on Amp plates. Patch again after 12h culture. Wait 12h but no colony found.
2. Extract plasmid from the inocula. recut the plasmid and get a clear line. Then try to link recA and recN onto the plasmid. 16°overnight.
1. Repeat the experiment of pET28a promoter cutting.
2. Eletrotransform the linking product into DH5a cells and culture them on Amp plates. Patch again after 12h culture.
NO COLONY FOUND!!!!!
Western Blot
1. Transfer the pET32 plasmid into E.coli BL21.
2. Culture the pET32a-BL21 E.coli and the trkD-pET32a-BL21 E.coli with 5ml LB medium.
3. Put 3ml culture of the pET32a-BL21 E.coli and the trkD-pET32a-BL21 E.coli into 100ml CsCl-LB solution (CsCl content: 0.4g/100ml LB) respectively. Two hours later, their respective OD values are 0.35 and 0.53. Then induce them with 500μl IPTG(concentration: 0.1mol/L). Four and a half hours later, their respective OD values are 1.03 and 1.14. Then keep them still in the 37℃ incubator for 4 hours. Centrifugate the culture to collect the E.coli, then wash them with PBS for two times. In the end, suspend the E.coli with 20ml ddH2O respectively. Then send the two samples of the pET32a-BL21 E.coli and the trkD-pET32a-BL21 E.coli to China National Analytical Center, Guangzhou for the analysis of Cs element.
Western Blot
1. Transform pSB1C3, pSB1A3, pSB1T3 and BBa_J04450 to DH5α.
2. Transform cheZ-pET-28a to strains whose cheZ gene is knocked out, and smear them on the medium with IPTG line to test chemotaxis.
1. Double digest gfp-pUC18 and recA-cheZ(without terminator) with EcoRI and PstI in the 30μl system. Ligate gfp-pUC18 with recA-cheZ(without terminator) overnight. Electrotransform the ligation product into E.coli DH5α. Smear them on the solid medium containing Ampicillin.
2. Double digest pET28a with BamHI and BglII, and then ligate itself with T4 ligase overnight.
3. Use colony PCR to detect whether recA-gfp-pUC18-infusion is successfully constructed. The result indicates that it fails.
4. Sequence the only grown E.coli in yesterday which contain recA-gfp-pUC18-infusion.
Do PCR on recN and trkD (for biobrick assembling) with KOD plus® polymerase, but failed.
1. Electrotransform the self-ligation product of pET28a into DH5α, and smear them on the solid medium.
2. Result of colony PCR shows that I fail to construct recA-GFP-pUC18 in-fusion.
1. Do gradient PCR to clone recN and trkD (for biobrick assembling) with Go Taq®, but failed again.
2. Try to use cheZ-pET-28a to test chemotaxis again.
Western Blot
1. Culture the E.coli with cheZ-gfp-pUC18 and trkD-gfp-pUC18 with 5ml LB medium overnight.
2. Expand the culture with 100ml LB medium containing ampicillin. Two hours later, both of their OD values are 0.54. Then divide the 100ml culture into two bottles of 50ml, and induce one of the two with IPTG, keeping the other as the control. Four hours later, observe the expression of EGFP with confocal laser scanning microscopy. The result is that the experiment group is brighter than the control group.
1. Use Go Taq® to PCR recN again, but failed again.
2. Collect plasmid pSB1C3 to test the length of it.
1. Digest plasmid pSB1C3 with EcoRI, SpeI, XbaI and PstI, respectively, to test the plasmid. (1 and 6 is 1Kb marker, 2~5 are pSB1C3 digested by EcoRI, SpeI, XbaI and PstI, respectively).
2. Use new template bacterial to PCR recN and trkD (for biobrick assembling) with Go Taq®, and succeed in time; then amplifying cloning the genes.
Western Blot
1. Continue Zhao's experiment to pick the single colony of infusion recA-gfp-pUC18-DH5α, pET28a-T7 promoter knock-out-DH5α, and recA-cheZ(no ter)-gfp-pU18-DH5α. When the colony grows up, conduct the colony PCR of infusion recA-gfp-pUC18-DH5α and recA-cheZ(no ter)-gfp-pU18-DH5α, but there are no expected results. Extract the plasmids of pET28a-T7 promoter knock-out-DH5α, and then digest them with SmaI and XbaI, the system is as following: ddH2O: 15μl Buffer 4: 3μl Plasmid: 10μl SmaI: 1μl XbaI: 1μl In the end, there is also no expected result.
Double digest recN, trkD (for biobrick assembling) and plasmid pSB1C3 with XbaI and SpeI, and then ligate the genes to the plasmid. (1 is 1Kb marker, 2 is recN, 3 is trkD, 4 is pSB1C3).
Double digest recA, recN and plasmid pUC18-EGFP with XbaI and EcoRI-HF, and then ligate the genes to the plasmid.
1. Receive the Cs analysis report of last experiment, and the results are as following: Control: 23.9mg/Kg (sample weight: about 20g) Experiment: 32.8mg/Kg (sample weight: about 20g).
2. Repeat the experiments from 2011-9-4 to 2011-9-6, but the original Cs concentration is 0.05g CsCl/100ml LB. Induce the experiment group with IPTG at 18℃ overnight. Then keep them still in the 37℃ incubator for 6 hours. Collect the E.coli for analysis.
Transform recA-pUC18-EGFP and recN-pUC18-EGFP to DH5α.
1. Find out that nalidixic acid or mitomycin C have the same effect on Promoter recA or recN.
2. Plan to ligate recA with trkD if the ligation of recN and trkD cannot be accomplished.
3. T7 promoter of pET28a has been cut off.
Do Clony PCR on recN-pSB1C3, trkD-pSB1C3, recA-pUC18-EGFP and recN-pUC18-EGFP to test the ligation.
1. Transform cheZ-pET-28a to strain whose cheZ gene is knocked out.
2. Collect plasmid recN-pSB1C3, trkD-pSB1C3, recA-pUC18-EGFP and recN-pUC18-EGFP and double digest the first one with EcoRI and SpeI, second one with EcoRI and XbaI, third one and forth one with EcoRI and HindIII, which shows the false-positive result of recN-pSB1C3, trkD-pSB1C3.
Western Blot
Re-ligate recN and trkD to pSB1C3 and then transform them into DH5α. After culture over night, do clony PCR to test whether the ligation is successful, and both are successful. (1 is 1Kb marker, 2 is recN, 3 is trkD, 4 is pSB1C3).
1. Receive the Cs analysis report of last experiment, and the results are as following: Control: 5.58mg/Kg (sample weight: 23.22g). Experiment: 5.38mg/Kg (sample weight: 21.17g).
2. Repeat the experiments from 2011-9-4 to 2011-9-6, but the original Cs concentration is 0.6g CsCl/100ml LB. Induce the experiment group with IPTG at 25℃ overnight. Then keep them still in the 37℃ incubator for 6 hours. Collect the E.coli for analysis.
3. Receive the Cs analysis report of last experiment, and the results are as following: Control: 112mg/Kg (sample weight: 19.20g) Experiment: 77.9mg/Kg (sample weight: 20.94g).
Use Go Taq® to PCR trkD (for ligation with recN-pUC18-EGFP).
Western Blot Succeed
1. Amplifying cloning of trkD (for ligation with recN-pUC18-EGFP) with PrimeStar®.
2. Double digest pSB1C3 with EcoRI and PstI.
3. Gradient PCR genes (recA1, recN2, cheZ3, cheZ'4, trkD5, trkD'6, EGFP7) for biobrick assembling with Go Taq® to search proper condition of PCR.
4. Double digest trkD (for ligation with recN-pUC18-EGFP) with XbaI and HindIII.
1. Amplifying cloning of genes for biobrick assembling with PrimeStar®.
2. Double digest genes for biobrick assembling with EcoRI and PstI, and then ligate them with pSB1C3 digested by same restrictive enzymes. (1~7 are pSB1C3, recA, recN, cheZ, cheZ', trkD, EGFP).
Repeat Western Blot
Transform products of ligation into DH5α, and the results shows the failure of the ligation. And then do this ligation again with new enzymes.
1. Zhao has constructed the plasmid recA-gfp-pET28a(T7 knock-out) and recA-gfp-pET28a(T7 knock-out), So I cut of the gfp gene from the two kinds of plasmid.
2. Try to ligate trkD-gfp or only trkD into the two kinds of plasmid. Although some E.coli could grow on the culture containing antibiotic, the results of colony PCR are not expected. In the end, all tries to ligate trkD-gfp or only trkD into the plasmids recA-gfp-pET28a (T7 knock-out) and recA-gfp-pET28a (T7 knock-out) have failed.
Re-clone all the genes again and digest them again, then ligate them with plasmid again to test whether the ligation is successful. Do transformation. (1~8 are recA, recN, EGFP, pSB1C3, cheZ, cheZ', trkD, trkD', respecitvely).
Inoculate two strains: recA-GFP-pET28a(T7 knock-out)-BL21 and recN-GFP-pET28a(T7 knock-out)-BL21.
Double digest genes for biobrick assembling with XbaI and SpeI, and then ligate them with pSB1C3 digested by same restrictive enzymes. Do transformation. (1~8 are EGFP, trkD, pSB1C3, recA, recN, cheZ, trkD', cheZ', respectively).
Exploring E.coli movement induced by NAL or UV.
1. Mix 1.4 ml each inocula with 70ml LB, then place them into the 37°220rpm shaker. Separate every 10 ml inocula into an aseptic conical flask, and induce with a specific concentration of NAL. There are 6 experiment groups and one control group. Place all the conical flasks into shaker at 37°220rpm for 4h. Then add 200ul to every hole of a microplate and exam the fluorescence intensity by the Microplate Reader.
2. Inoculate the same stains as last night.
1. Digest recA-gfp-pET28a (T7 knock-out) and gene trkD with XbaI and HindIII respectively, and then to ligate them together.
2. Electrotransform the ligation product into E.coli DH5α, and smear them on the solid medium containing Kanamycin. After the colony grows up, pick up the single colony to smear on another medium. After 6 hours' culture, the colony grows up, and then conduct the colony PCR to copy the gene trkD. The result indicates that I have successfully construct the recA-trkD-pET28a (T7 knock-out) plasmid.
1. Mix 3.4 ml each inocula with 170ml LB, then place them into the 37°220rpm shaker. Separate every 10 ml inocula into an aseptic conical flask, and induce with a specific concentration of NAL. There are 6 experiment groups and one control group. Place all the conical flasks into shaker at 37°220rpm for 4h. Then regulate OD value of the inocula in every conical flask to 2.023 to 2.033. The remaining 100ml incula was separated to 20 centrifuge tubes. 5ml inocula in every tube. Place all the tubes under the UV light. Take out one tube every 10min and mark the time on the tube. Then add 200ul to every hole of a microplate and exam the fluorescence intensity by the Microplate Reader.
2. Inoculate three stains: the same stains as last night and the strain of BL21.
Mix 1.4 ml each inocula with 70ml LB, then place them into the 37°220rpm shaker. Separate every 10 ml inocula into an aseptic conical flask, and induce with a specific
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