-culture the competence cell
-test the experiment kits

-Molecular cloning for Bba_J06602, BBa_B0012, Bba_K592024, Bba_I13453, Bba_B0010
-Tried to reverse pBAD(BBa_I13453)
-preparation of the target bio-brick

-Ligation: flpe-BFP, loxP-pBAD, B0010-loxP, eGFP-B0015, FRT-pBAD
-Preservation of the plasmid pAUR135 in E.coli; Transformation of the commercial plasmid pAUR135(from TAKARA, Item No.D3604); select monoclonal colony and extract plasmid(pAUR135) from the culture.
-Prepared Yeast Extract Peptone Dextrose(YPD) Medium

-Patch: strain: S.cerevisiae W303; medium: YPD agar

-Extract the genome of the budding yeast as the template used in the subsequence PCR, yeast stains was Saccharomyces cerevisiae laboratory strain W303 (haploid), and the extraction procedure is based on the fungal genome extraction kit(Omega Bio-Tec).
-Poor yield, partly because the yeast concentration in the suspension is low for genome extraction. The time for yeast incubation should be prolong for extraction next time.

-ligation: pBAD-eGFP- B0010;pBAD-EFP- B0010;PBAD-mCherry- B0010.
-induction and test the intensity of fluorescence
-HOWEVER our results went bad
-Extract genome DNA from W303 with yeast genome extraction kit(Omega Bio-Tec).

-Acquire homologous franking sequence for target integrating site with PCR procedure, using the extracted genome from W303 as a template.
-Ultraviolet exposure result of the identification gel shows nonspecific amplification. We plan to employ nested PCR.
-Preservation of W303 strain.
-Extract genome DNA from W303 with yeast genome extraction kit(Omega Bio-Tec).

-Employ nest PCR to acquire homologous sequence and M phase Promoter sequence using W303 genome.
-Ultraviolet exposure result of the identification gel shows no target band.

-Employ nest PCR and gradient anneal temperature from 50-60℃ to define the best anneal temperature.
-Anneal at 60℃ should be better.

-The ligation of pBAD-FR, pBAD-flpe is successful, we tried to insert Cre segment to pSB1A2
-Employ nest PCR and gradient anneal temperature from 56-66℃ to define the best anneal temperature.
-The most appropriate anneal temperature should be 65℃.
-Extract genome DNA from W303 with yeast genome extraction kit(Omega Bio-Tec).

-Ligation again: pBAD-mCherry-B0010, pBAD-BFP-B0010, pBAD-eGFP-B0015
-induction and test the intensity of fluorescence
-Again, we ligated pBAD with FRT

-We designed qPCR experiments to test whether our system work.

-We ordered our IDT gBlocks products.
-We tested whether our ligation products were corrected by sequence and enzyme digestion, and we found that BBa_B0010 in distribution plate was a fake one!
-Acquire M phase promoter sequence with GenStar 2x Taq PCR Star(Mix with Loading Dye) using W303 genome.
-Clean up the PCR product(M phase promoter nest sequence).
-Mutation of the plasmid pAUR135.( Muta-direct™ Kit from SBS Genetech Co.,Ltd. SDM-15).

-Add RBS and ssrA(strong) to mCherry by PCR and Extract PCR products by using Gel Extraction Kit.
-Double digestion of RBS-mc-ssrA(strong) PCR product and pSB1C3(r) with Spe I and EcoR I. Extract double digestion products by using Gel Extraction Kit.
-Ligation of RBS-mc-ssrA(strong).
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-We conducted digestion identification and sequencing. No sequencing results were obtained, and the sizes of digestion products were incorrect!
-Sequencing result of pAUR135 mutant: pAUR135 mutant have A1041C mutation in CDS of AurR gene, which result in the elimination of Pst I restriction site.
-Enzyme digestion with FD Pst I (Takara)
-Extract the product(pAUR135 backbone with pst I sticky end) of 5000+bp
-End-filling with S1 nuclease.
-Ligation with T4 DNA polymerase.

-Pick single colonies of RBS-mc-ssrA(strong), and streak them onto another plate to expand culture.
-Perform colony PCR of RBS-mc-ssrA(strong).
-Pick correct colonies of RBS-mc-ssrA(strong), and inoculate a culture of 4 ml LB medium containing chloromycetin.
-We noted that BBa_B0010(fake) can ligate with our parts, and thus any part ligated with it must be reconstructed!
-We began to reconstruct our parts, and some of our parts, like Cre and eGFP run out. We amplified BBa_B1006 and BBa_B0015 in DH5a.
-Transform 5ul T4 ligation product (Jul.15) into 50ul DH5α.
-Select monoclonal colony and extract plasmid (pAUR135 circular backbone without pst I).
-Checkout of the removal of Pst I enzyme site.

-Use Plasmid Miniprep Kit to isolate mc-ssrA(strong) from the cultures.
-We received our booked IDT gBlocks products arrived!
-We changed our circuits by replacing some devide parts with composite parts. we converted BFP to BBa_E0422(ECFP), converted eGFP to E0840 (GFP with terminator), such changes can accelerate our work
-Double enzyme digestion (HF，NEB) with EcoR I and Sac I.
-Gel extraction (Digestion product with EcoR I and Sac I sticky end) With Tiangen gel extraction kit.
-Anneal of MCS oligonucleotide.
-Ligation with double digested backbone and MCS anneal product.
-Transformation with ligation product.
-no colony found on the plate
-Double enzyme digestion did not work, as enzyme Sac I has very low(~20%) efficiency when the space between the two enzyme sites is very close(2bp in this experiment).

-Add RTS sequences (loxp) to the flanks of RBS-mC-ssrA(strong)-1 by PCR.
-Extract PCR products by using Gel Extraction Kit.
-Double digestion of loxp-mcss-1 and B0015 (B0015 digest with Xba I and EcoR I, Fragments digest with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-Ligation of loxp-mcss-T.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Later sequence result shows the template we used, RBS-mC-ssrA(strong)-1, is not correct. So the clones made by this day are not correct too.
-We tested and confirmed that mCherry, E0422, E0840, Cre and B1006. They were all corrected.
-We ligated E0840 with loxP(reverse) and mCherry with B1006. But nearly no clone grown.
-We obtained some clones with ligation products of mCherry-B1006
-The first part, BBa_K1641213, was successfully constructed!
-Parts construction
-Adjust double enzyme digestion into two single digestions, first Sac I then EcoR I.
-MSC modification fails.
-Change MSC modification primer using Kpn I and EcoR I as sticky end.
-Double enzyme digestion with Kpn I and EcoR I and then ligate with MCS annealed product.
-Transformation.
-Extract plasmid: pAUR135-KE.
-Sequencing result shows incorrect ligation.

-Add RTS sequences (loxp) to the flanks of RBS-mC-ssrA(strong)-2 by PCR. Extract PCR products by using Gel Extraction Kit.
-Ligated E0840 with loxP and pBAd-flpe-ECFP. We tried several methods of ligation. Preliminary "2A" assembly mthod was developed. However, colony PCR showed high false positive rate of these clones.

-Double digestion of loxp-mcss and B0015. (B0015/B1006 digested with Xba I and EcoR I, Fragments digested with Spe I and EcoR I).Extract double digestion products by using Gel Extraction Kit.
-Ligation of loxp-mcss-T.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Pick single colonies of loxp-mcss-T, and streak them onto another plate to expand culture.

-Perform colony PCR of loxp-mcss-T and record the correct colonies.
-Pick correct colonies of loxp-mcss-T, and inoculate a culture of 4 ml LB medium containing chloromycetin.
-Use Plasmid Miniprep Kit to isolate loxp-mcss-T from the cultures.
-We used PCR to reconstruct RBS-Cre-ssrA, and inserted this fragment into pSB backbone.
-All clones grown well! But somebody placed our enzymes out of refrigerator overnight. We went crazy! So our process was delayed.
-We placed the gBlocks products into pSB backbones. Most of them were successful. Also we gained some correct clones of loxP-GFP(BBa_K16416209).
-Ligations: Cre with mCherry and K1641206 with flpe-ECFP
-pAUR135-MCS modification
-Double enzyme digestion with Kpn I and EcoR I and then ligate with MCS annealed product.
-Transformation;Extract plasmid: pAUR135-MCS-KE
-Clone biobrick:KRE9UTR;GFP-PEST191;SV40NLS;Venus YFP
-Patch:bxb1gp35 RFC25optimized-puc19
-Transformation and Plasmid Preparation-MCS-KE
-Sequencing confirmed.

-Again, we tried to ligate the short intermediates with the long parts:pBAD(reverse) with FPs, etc.
-We successfully ligated pBAD(reverse) with flpe-ECFP.
-We used endonuclease and sequencing skills to test the correctness of ligation
-Clone cyclic promoters;PCR :YMR013C/Cln3p/Met16p from yeast genome add attB/P sites, prefix and suffix
-transformation and plasmid preparation:MCS-KE
-Assemble:Pgal+kozak-1C3;bxb1gp35+SV40NLS-1C3;;Pbad-RBS-1C3
-Double digestion, ligation and transformation
-Mutation:MCS-KE to RFC25 optimized
-Substrate digested via Fast Digest-Dpn I.
-product transformed in TOP10 50, Amp

-Double digestion of loxp-mc-1, loxp-mc-2, B0015 and CG, CGS, B1006 (B0015/B1006 digested with Xba I and EcoR I. Fragments digested with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-Ligation of loxp-mc-1-T, loxp-mc-2-T and CGT, CGST.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Use Plasmid Miniprep Kit to isolate I0500 from the cultures.
-We ligated pBAD(forward) with other parts contains FPs
-We test whether pBAD and FPs can worked, but we met high false positive rates.
-We found that the ECFP sequence of our BBa_K1641209 sample was reverse! Oh my God!
-Clone biobrick:ECFP;EYFP;mRFP1
-PCR:reversed mRFP1;reversed KRE9UTR
-Plasmid preparation: pAUR135 RFC25 optimized
-Assemble:reversed KRE9UTR+reversed mRFP1;eCFP+KRE9UTR
-Double digestion, ligation and transformation
-Add sequence via PCR: prefix-RBS-eCFP-terminatior-frt;loxP-RBS-Cre-ssrA;prefix-RBS-GFP-Terminator-loxp;prefix-ECFP-frt-suffix;prefix-loxP-Cre-suffix;prefix-GFP-loxp-suffix
-Assemble:Met16p-eCFP;YMR013C-eCFP;Loxp-cre+gfp-loxp;frt-flpe-cfp-frt
-Transformation.
-Plasmid preparation.

-Double digestion of dre, vika, scre, vcre, pSB1C3(r) with Spe I and EcoR I. Extract double digestion products by using Gel Extraction Kit.
-Ligation of dre, vika, scre, vcre and pSB1C3(r).
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Pick single colonies of dre(1c3), vika(1c3), vcre(1c3), scre(1c3), and streak them onto another plate to expand culture.
-Perform colony PCR of dre(1c3), vika(1c3), vcre(1c3), scre(1c3) and record the correct colonies.
-Pick correct colonies of dre(1c3), vika(1c3), vcre(1c3), scre(1c3), and inoculate a culture of 3 ml LB medium containing chloromycetin
-Add RTS sequences(loxp) to the flanks of RBS-mC-ssrA(strong) by PCR. Electrophoresis result didn’t shows correct bands.
-Use Plasmid Miniprep Kit to isolate dre(1c3), vika(1c3), vcre(1c3), scre(1c3) from the cultures.
-We tried to amplify BBa_K1641203, BBa_K1641201, BBa_K1641209
-We ligated mCherry with BBa_K1641208.
-Assemble:reversed KRE9UTR+reversed mRFP1;eCFP+KRE9UTR
-Restriction analysis:loxp-cre-gfp-loxp
-Assemble:YFP-KRE9UTR;reversed KRE9UTR+reversed mRFP1
-Sequencing:loxp-cre-gfp-loxp

-Prepare RBS-Flpe-fc by PCR.
-Use Plasmid Miniprep Kit to isolate CG, CGS, B0015, B1006, I0500 from the cultures.
-Add RTS sequences(loxp) to the flanks of RBS-mC-ssrA(strong) by PCR. Electrophoresis result didn’t shows correct bands.
-Add RTS sequences(FRT, Rox, SloxpM1, Vloxp, vox) to the flanks of RBS-mC-ssrA(strong) by PCR. Electrophoresis result didn’t shows correct bands.
-We had many colony PCR experiments to find a correct clone, we did.
-We eventually had a real BBa_K1641209. Also we have correct BBa_1641203 and BBa_K1641201. So we can use these parts to construct the Circuit2, which was improved on the basis of Circuit1.
-clone and assemble:loxp-cre-gfp-loxp;frt-flpe-cfp-frt
-sequencing inconsistant

-Double digestion of PCG-1, PCGS-1, B1006. (B1006 digested with Xba I and EcoR I, fragment digested with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-Ligation of PCGT, PCGST.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Perform 20ul system PCR to test if the primers of FRT, Rox, SLM, VL, vox work.
-Add RTS sequences to the flanks of FRT, Rox, SLM, VL, vox by PCR. Extract PCR products by using Gel Extraction Kit.
-Double digestion of FRT, Rox, SLM, VL, vox and pSB1C3(r) with EcoR I and Pst I. Extract double digestion products by using Gel Extraction Kit.
-Ligation of FRT, Rox, SLM, VL, vox.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Use Plasmid Miniprep Kit to isolate PCGT-1, PCGST-1, pSB3k3 from the cultures.
-Double digestion of PCGT-1, PCGST-1 and pSB3k3. Extract double digestion products by using Gel Extraction Kit.
-Ligation of PCGT-1(3k3), PCGST-1(3k3).
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+kan.
-Perform colony PCR of FRT-mcsm, Rox-mcsm, VL-mcsm, vox-mcsm.
-Pick correct colonies of FRT-mcsm, Rox-mcsm, VL-mcsm, vox-mcsm, and inoculate a culture of 4 ml LB medium containing chloromycetin.
-Perform colony PCR of RBS-mc-ssrA(strong).
-Pick correct colonies of RBS-mc-ssrA(strong), and inoculate a culture of 4 ml LB medium containing chloromycetin.
-We successfully ligated pBAD(reverse) with flpe-ECFP and we also got correct BBa_K1641214.
-We tried to induce BBa_K1641214 in DH5a and Top10 strains with L-arabinose in LB broth, then test the intensity of fluorescence

-Use Plasmid Miniprep Kit to isolate FRT-mcsm-3, Rox-mcsm-3, SLM-mcsm-4, VL-mcsm-4, vox-mcsm-2,VGC-3, PCGT(3k3), PCGST(3k3), mc-ss-2 from the cultures.
-Double digestion of ScGS-1, VGS-3, VcGS-4, FGS-1, DGS-1 and I0500. Extract double digestion products by using Gel Extraction Kit.
-Ligation of PDGS, PFGS, PVcGS, PVGS, PScGS.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Perform colony PCR of PDGS, PFGS, PScGS, PVcGS.
-Pick correct colonies of PDGS, PFGS, PScGS, PVcGS, and inoculate a culture of 4 ml LB medium containing chloromycetin.
-Double digestion of RT-mcsm-3, Rox-mcsm-3, SLM-mcsm-4, VL-mcsm-4, vox-mcsm-2 and B0015. (B0015 digested with Xba I and EcoR I, fragment digested with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-Ligation of FRT-mcsm-T, Rox-mcsm-T, SLM-mcsm, VL-mcsm-T.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Use Plasmid Miniprep Kit to isolate PDGS-2, PFGS-5, PVcGS-1, PVGS-4, p23101, B0015, loxp-mcsm-Inv-rep from the cultures.
-Double digestion of PDGS-2, PFGS-5, PVcGS-1, PVGS-4 and B1006. (B1006 digested with Xba I and EcoR I, fragment digest with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-Ligation of PDGST, PFGST, PScGST, PVcGST.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Transform PCGT with loxp-mcsm-Inv-rep, PCGST with loxp-mcsm-Inv-rep, plate the transformed cells on LB+kan+cm.

-Use Plasmid Miniprep Kit to isolate FRT-mcsm-T, Rox-mcsm-T, SLM-mcsm, VL-mcsm-T from the cultures.
-Extract double digestion products by using Gel Extraction Kit.
-Ligation of P-FRT-mcsm-T(FRT-reporter), P-Rox-mcsm-T(Rox-reporter), P-SLM-mcsm(SLM-reporter), P-VL-mcsm-T(VL-reporter).
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-Use Plasmid Miniprep Kit to isolate PDGST-5, PFGST-1, PScGST-4, PVcGST-1, VGS-4, VGS-5 from the cultures.
-Double digestion of PDGST-5, PFGST-1, PScGST-4, PVcGST-1 and pSB3K3. Extract double digestion products by using Gel Extraction Kit.
-Ligation of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3).
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+kan.
-pcr (add sequence):prefix-RBS-flpe-FC, FC-Mcherry-T7-frt-suffix from biobrick
-overlap pcr:RBS-flpe-FC-Mcherry

-Pick single colonies of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3), and streak them onto another plate to expand culture.
-Perform colony PCR of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3). Colony No. 2, 3, 4, 6 of PDGST-5(3k3), No. 2, 3, 4, 5 of PFGST-1(3k3), No. 1, 2, 3, 4, 5, 6 of PScGST-4(3k3), No. 3, 4, 5, 6 of PVcGST-1(3k3) are correct.
-Pick correct colonies of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3), and inoculate a culture of 4 ml LB medium containing kanamycin.
-Use Plasmid Miniprep Kit to isolate PDGST-5-2, PFGST-1-2, PScGST-4-1, PVcGST-1-4 from the cultures.
-Double digestion of PDGST-5-2, PFGST-1-2, PScGST-4-1, PVcGST-1-4 and pSB1K3 with EcoR I and Pst I. Extract double digestion products by using Gel Extraction Kit.
-Ligation of PDGST-5-2(1k3), PFGST-1-2(1k3), PScGST-4-1(1k3), PVcGST-1-4(1k3).
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+kan.
-Transform PDGST-2(3k3) with Rox-repoter, PFGST-4(3k3) with FRT-reporter, PScGST-1(3k3) with SLM-repoter, PVcGST-2(3k3) with vox-repoter, plate the transformed cells on LB+kan+cm.
-We detected ECFP in M9 broth!
-BBa_K1641225 correctly constructed!
-pcr (add sequence):prefix-flpe-FC, FC-Mcherry-T7TE-frt-suffix from biobrick
-overlap pcr:flpe-FC-Mcherry;overlap pcr worked
-assemble:Pbad-frt-RBS-flpe-FC-Mcherry-T7TE-frt in pSB1A2

-Use Plasmid Miniprep Kit to isolate cgs, vgs, gp38 from the cultures.
-Pick single colonies of PDGST-4(1k3), PFGST-4(1k3), PScGST-4(1k3), PVcGST-1(1k3) , and streak them onto another plate to expand culture.
-Perform colony PCR of PDGST-4(1k3), PFGST-4(1k3), PScGST-4(1k3), PVcGST-1(1k3). Colony No. 1, 2, 3, 4, 5, 6 of PDGST-4(1k3), No. 2, 4, 6, 7 of PFGST-4(1k3), No. 1, 2, 3, 4, 5, 6 of PScGST-4(1k3), No. 1, 2, 3, 4, 5, 6 of PVcGST-1(1k3) are correct.
-Pick correct colonies of PDGST-4(1k3), PFGST-4(1k3), PScGST-4(1k3), PVcGST-1(1k3), and inoculate a culture of 4 ml LB medium containing kanamycin.
-Use Plasmid Miniprep Kit to isolate PDGST(1k3), PFGST(1k3), PScGST(1k3), PVcGST(1k3), Test-mc-T, Test-rep, p23101, I0500, B1006 from the cultures.
-Double digestion of FRT-mcsm-T, Rox-mcsm-T, SLM-mcsm-T, VL-mcsm-T (Spe I and Pst I) and p23101(Xba I and Pst I). Extract double digestion products by using Gel Extraction Kit.
-Ligation of p-FRT-mcsm-T, p-Rox-mcsm-T, p-SLM-mcsm-T and p-VL-mcsm-T.
-Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-We conducted fluorescence measurements to confirmed whether BBa_K1641225 can work. We still tried to construct a full plasmid in Circuit 2.
-pcr (add sequence):prefix-loxP-RBS-Cre-FC, FC-eGFP-T7TE-loxP-suffix from biobrick
-overlap pcr:loxP-RBS-Cre-FC-eGFP-T7TE-loxP;overlap pcr worked
-assemble:Pbad-frt-RBS-flpe-FC-Mcherry-T7TE-frt-loxP-RBS-Cre-FC-eGFP-T7TE-loxP in pSB1A2