Team:Wageningen UR/Notebook/toggleswitch
Week 1 (2nd-7th June 2016)
Some of important biobricks for constructing toggle switch were transformed for the iGEM 2016 kits, including repressors BBa_P0440, BBa_P0412, inducible promoters BBa_R0040, BBa_R0010 and reporter genes BBa_K515005, BBa_K1365020. Breed conversation of each biobrick had been made.
Week 2 (29th- 30th June , 2016)
Purified BBa_K1491009 plasmid (132 ng/μL) from breed conservation.
Week 3 (4th-8th July, 2016)
Firstly, purified plasmid of BBa_P0440, BBa_P0412, BBa_R0040, BBa_R0010 (60.7 ng/μL, 50.7 ng/μL, 66.3ng/μL, 26.3ng/μL), secondly, transformed parts from 2016 kits including, BBa_C0040, BBa_C0012, BBa_B0015, and BBa_K1365020. Picked up colony from the plates, cultured and purified plasmid of each part (207.5 ng/μL, 140.6 ng/μL, 96.1 ng/μL, 146.3 ng/μL). Last, tried to use PCR to amplify iGEM vectors of each resistance by using the linearized backbones as template, but it failed.
Week 3 (4th-8th July, 2016)
Firstly, purified plasmid of BBa_P0440, BBa_P0412, BBa_R0040, BBa_R0010 (60.7 ng/μL, 50.7 ng/μL, 66.3 ng/μL, 26.3 ng/μL), secondly, transformed parts from 2016 kits including, BBa_C0040, BBa_C0012, BBa_B0015, and BBa_K1365020. Picked up colony from the plates, cultured and purified plasmid of each part (207.5 ng/μL, 140.6 ng/μL, 96.1 ng/μL, 146.3 ng/μL). Last, tried to use PCR to amplify iGEM vectors of each resistance by using the linearized backbones as template, but it failed.
Week 4 (11th-16th July, 2016)
Firstly, I tried to make vectors of each resistance again. This time, I transformed 6 kind of backbones that contains RFP generator from 2016 kits and picked up colony from the plates, cultured and purified plasmid of each part. Then digested them with EcroRI and PstI overnight and did gel purification to recycle backbones fragments. Final concentration of each vector (pSB1A3 17.2 ng/μL, pSB1A5 21 ng/μL, pSB1C3 18.3 ng/μL, pSB1C5 45 ng/μL, pSB1K3 35.9 ng/μL, pSB1LK5 12.6 ng/μL). I got 4 parts from iGEM part registry BBa_K215108, BBa_K215107, BBa_K554007, BBa_K554102. I did plate streaking and selected signal colony from the plate, then purified plasmid of each part (BBa_K215108 46.3 ng/μL, BBa_K215107 51.5 ng/μL)(Fig.1).
Week 5 (19th-24th July, 2016)
Firstly, I sent two secretion system BBa_K215108, BBa_K215107 to sequencing by using verification primer and got part of correct sequencing results. Secondly, I first digested BBa_P0440, BBa_P0412 with EcroRI and SpeI, BBa_K1491009 with XbaI and PstI and did 3A assembly of BBa_P0440- BBa_K1491009 and BBa_P0412- BBa_K1491009 with pSB1A3 vector separately. Then transformed ligation product into DH5α cell cultured overnight. Picked up colony from the plates and colony PCR to amplify the inserted part (failed). I also tried to add degradation tag BBa_M0050 to the C terminal of mRFP BBa_K1491009 and sfGFP BBa_K515005 by using PCR. The annealing temperature of both PCR reaction were 64℃, 1.5 min. Then did PCR product clean-up and each part BBa_K1491009+BBa_M0050 (145 ng/ μL), BBa_K515005+ BBa_M0050(318.6ng/ μL), BBa_K1365020+ BBa_M0050(239.2 ng/ μL). Then I digested each part with EcoRI and PstI 1 hour, 37 ℃ and ligated them with pSB1C3 vector 2 hour 16℃.
Week 6 (26th July -2nd August, 2016)
Firstly, I tried to use PCR amplify (annealing temperature 60℃) each hybrid promoter that we ordered from IDT. But only BBa_K1913026 had been amplified correctly (Fig.2) after PCR-clean-up (102.1 ng/ μL), I digested it with EcoRI and PstI 1 hour and ligated them with pSB1C3 vector 2 hour. And transformed it into DH5α cell and got the plasmid finally (40 ng/ μL). In order to get other hybrid promoter by using PCR, I diluted DNA solution of IDT 10 times and used it to do PCR again twice, but the bands were still weak(Fig.3). Secondly, I transformed BBa_K1491009+BBa_M0050, BBa_K515005+ BBa_M0050 and picked up colony from the plates and checked the correct inserted part by using PCR. And then culture them over night and purified plasmids (76 ng/ μL, 149.6 ng/ μL). And the sequencing results of these two construction were correct. Last, after I got the plasmid of BBa_K1491009+BBa_M0050, I digest digested it with EcoRI and SpeI 1 hour as well as BBa_P0440 and BBa_P0412 with XbaI and PstI 1hour. and ligated them with pSB1A3 vector 2 hour. Then I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.4). After plasmid purification of these two construction (mRFP+P0440 117.6 ng/ μL, mRFP+P0412 93.2ng/ μL). And also the sequencing results of these two construction were both correct. In addition, I cultured riboswitch strains contain BBa_K1913008 and BBa_K1913009.
Week 7 (3rd -9th August, 2016)
Firstly, I tried to PCR amplify hybrid promoter again by increasing the annealing temperature to 66℃. And I did gel purification of each hybrid promoter PCR products(Fig.5). And then, I digested it with EcoRI and PstI 1 hour and ligated them with pSB1C3 vector 2 hour. After transformation of each hybrid promoters into DH5α cell, I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain. Finally, I got plasmid of each hybrid promoter (BBa_K1913025:42.7 ng/ μL, BBa_K1913022:44.3 ng/ μL, BBa_K1913024 :44 ng/ μL, BBa_K1913023:37.5 ng/ μL). Secondly, I digested hybrid promoter 1 and 2 (BBa_K1913025, BBa_K1913022) with EcoRI and SpeI as well as mRFP+P0440 with XbaI and PstI and ligated hybrid promoter 1,2 with mRFP+P0440 in pSB1K3. After transformation of these two constructions, I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.6). Then, after plasmid purification, (P1+mRFP+P0440:245.1 ng/ μL, P1+mRFP+P0440:56.1 ng/ μL) I sent them to sequencing and results are correct. In addition, at the same time, I tried to use PCR amplify riboswitch part BBa_K1913008 and BBa_K1913009 by using specific primers, but it all failed. Meanwhile, I tried different annealing temperatures setting, for example, from 66℃ to 72℃, 15s for extension.
Week 8 (10th-16th August, 2016)
Firstly, I digested hybrid promoter 3 and 5 (BBa_K1913026, BBa_K1913024) with EcoRI and SpeI as well as mRFP+P0412 with XbaI and PstI and ligated hybrid promoter 3,5 with mRFP+P0412 in pSB1K3. Then I transformed ligation product into DH5α cell cultured overnight. I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain, but ligation of hybrid promoter 3 with mRFP+P0412 failed(Fig.7). The sequencing result of P5+mRFP+P0412 was correct. Then, I digested hybrid promoter 4(BBa_K1913023) with EcoRI and SpeI as well as mRFP+P0440 with XbaI and PstI and ligated hybrid promoter 4 with mRFP+P0440 in pSB1K3. After I transformed ligation product into DH5α cell cultured overnight, I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.8). After plasmid purification (78.9 ng/μL) I sent it to sequencing, And its sequencing results were also correct. Thirdly, I tried to use PCR amplify two signal riboswitches BS-xpt and BS-yxjA (66℃ annealing temperature and 15s extension time) that were order from IDT. I got the correct band and did gel purification(Fig.9). Then I digested BS-yxjA with EcoRI and SpeI as well as BBa_P0440 and BBa_P0412 with XbaI and PstI and ligated hybrid promoter 1,2 with mRFP+P0440 in pSB1A3. I transformed ligation products into DH5α cell cultured overnight. I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.10). I purified the colony PCR products and sent them to sequencing, and its results were correct. In addition, I transformed BBa K215001 from 2016 iGEM kits and cultured overnight. And purified plasmid (92.6 ng/μL) after transformation.
Week 9 (17th-23th August, 2016)
Firstly, I tried to ligate hybrid promoter 3 with mRFP+P0412 in pSB1K3 again, and then transformed it and picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.11). After plasmid purification (102.1ng/μL) I sent it to sequencing, And its sequencing results were also correct. Secondly, I purified plasmid of BS-yxjA with BBa_P0440 and BBa_P0412 (52.4 ng/μL, 78.4 ng/μL) and I digested BBa_R0010 with EcoRI and SpeI as well as BS-yxjA- P0440 with XbaI and PstI and ligated them in pSB1A3. I transformed ligation products into DH5α cell cultured overnight then I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.11). After plasmid purification (102.1 ng/μL) I sent it to sequencing, And its sequencing results was also correct. Secondly, I design three primers that can bind to the middle of BBa_K215108. And sent these primers to sequence BBa_K215108. Then I got the correct sequencing results of the whole sequence of BBa_K215108. In addition, I tried to use PCR to add NehI restriction site on the both side of Chitinase A and B (BBa_K1913000, BBa_K1913001). The annealing temperature of both reaction were 63℃. Only ChiA had been amplified, but for ChiB it still could be amplified, even if I tried to used different annealing temperature or DNA polymerase. Then I digested ChiA and BBa_K215001 with NehI 1 hour, and ligate them for 2 hours. Then I transformed it into DH5α cell cultured overnight I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.12). However, the sequencing results of this construction were not correct. Next, I digested BBa_R0440 with EcoRI and SpeI as well as Bs-yxjA-P0412 with XbaI and PstI and ligated them in pSB1K3. Also, I digested R0010+BS-yxjA-P0440 with EcoRI and SpeI as well as P5+mRFP-P0412 with XbaI and PstI and ligated them in pSB1K3. After I transformed ligation product into DH5α cell cultured overnight, I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain(Fig.13). However, only toggle switch part got correct sequencing results, R0440+BS-yxjA-P0412 part (366 ng/μL) failed. Last, I tried to use PCR amplify ChiB with NehI restriction site (59℃ annealing temperature and 2min extension time), then I did gel purification to get the correct band. After digestion with NehI and ligation with BBa_K215001, I did transformation and I picked up colony from the plates and checked inserted parts by PCR, but they were not correct.
Week 10 (24th-30thAugust, 2016)
Firstly, I tried to digest R0010+BS-yxjA-P0440 with EcoRI and SpeI as well as P3+mRFP-P0440 with XbaI and PstI and ligated them in pSB1K3. After transformation of this composite into DH5α cell, I picked up colony from the plates and checked inserted parts by PCR and cultured the correct strain. I failed in the first time. Thus, I tried to picked up colony from the plates again, and I got the correct results this time(Fig.14). After I got the plasmid(187.45 ng/μL) and sent to sequencing, the sequencing results was correct. Secondly, I also transformed BBa_J23114, BBa_B0032 from the iGEM 2016 kits, and purified plasmid of each part (76.9ng/μL, 54 ng/μL). I also tried to transform ChiB with tag again, but it did not succeed. Next, I co-transformed toggle switch device with light sensor BBa_K1913034 and picked up the correct colony from the plate, and tested toggle switch device 1-light sensor in different LB medium with gradient guanine concentration(from 0 to 0.1mg/L) under 8h culture. But it did not work. Last, I also tried to ligate R0440+BS-yxjA-P0412 part with P1+mRFP-P0440, but it did not succeed.
Week 11 (31st August-6th September, 2016)
Firstly, I purified plasmid of BBa_B0034 and BBa_J23114(46 ng/μL, 86 ng/μL), then I digested them and did 3A assembly with pSB1C3. But the results were not correct. Next, I tried to use PCR amplify vitamin B12 riboswitch and vitamin B12 riboswitch new that was order from IDT. Then, I did gel purification and got two parts (37 ng/μL, 26.9 ng/μL). Next, I amplified BBa_K592016 and BBa_K215001 by PCR. BBa_K592016, then was digested and ligated with BBa_J23108 in pSB1C3, BBa_K215001 was sent to sequencing and it was correct. Also, I digested BBa_C0012 and BBa_C0040 and ligated them with vitamin B12 riboswitch part in pSB1A3, mRFP-P0440 part was also digested and ligated with BBa_J23108 and riboswitch BS-yxjA in pSB1K3. After I transformed ligation product into DH5α cell cultured overnight, I picked up colony the plates and checked inserted parts by PCR and cultured the correct strain. But the colony from BBa_J23108 –mRFP-P0440 plate was in white. The BBa_C0012 and BBa_C0040 with vitamin B12 riboswitch and BBa_J23108 with mRFP-P0440 are correct according to the gel electrophoresis. After these samples was sent to sequencing, only BBa_C0012 and BBa_C0040 with vitamin B12 was correct. BBa_J23108 wasn’t ligate with mRFP-P0440. Next, I removed the first RBS part from mRFP-P0412 and mRFP-P0440 by using PCR and did gel purification to purified these two parts (92.6ng/ μL, 106.2 ng/ μL). Then I tried to ligate each hybrid promoter with mRFP-P0412 and mRFP-P0440 without RBS. And transformed these ligation product and cultured them overnight. Last, I tried to ligate ChiA part with secretion tag and transformed it, but the length of the final products were still same as the previous results.
Week 12 (7th -13th September, 2016)
Firstly, I picked up colony from the hybrid promoter with new mRFP-P0412 and mRFP-P0440 plates and checked inserted parts by PCR(Fig.15), only P1, P2 P3 plus mRFP part were correct, P1 and P4 with mRFP-P0440 failed. Then I sent the PCR products to sequence and the results were correct except for P3 with mRFP-P0440. Then I tried to amplify ChiA with tag and got three bands that was over 2000 bp. I sent them to sequence, but the results are still not correct. Next, I transformed BBa_J23106 and BBa_K516032 from the 2016 kits, after cultured them overnight, I purified plasmid (76.8 ng/μL, 59.6 ng/μL), then I digested them and ligate BBa_K516032 with BBa_J23106 and riboswitch Bs-yxjA part and Bs-xpt part. After transformation of these ligation products, I picked up colony from the plates and checked inserted parts by PCR. But the results were not correct at the first time. Next, I tried to ligate BBa_J23114 and BBa_B0034 again, but the results were still not good. Then, I amplified mRFP with terminator by using PCR and digested them with XbaI and PstI. Then I ligated each hybrid promoter with mRFP with terminator and P1-mRFP-P0440, P4-mRFP-P0440, BBa_J23106-mRFP-P0440 again. After transformation of these ligation products, I picked up colony from the plates and checked inserted parts by PCR(Fig.16). Then I sent PCR products to sequence but only promoter 1 with mRFP was correct.(In the first time, because I sent some short band PCR products to sequence, I got the wrong results. So I did the whole process again, but finally I found the all longer band of PCR products in the first experiment were correct.) Next, I digested vitamin B12+C0040 and C0012 and terminator BBa_B0015 in pSB1K3. After transformation of these ligation products, I picked up colony from the plates and checked inserted parts by PCR. After I sent the PCR product to sequence, only the VB12+C0040+B0015 was correct. At the same time, I tried to ligate ChiA and secretion tag again, but it still failed.
Week 13 (14th-20th September, 2016)
Firstly, I tried to do the gel purification of VB12-C0012+B0015(25.6 ng/μL) and used it as template to amplify this part. After PCR product purification, I sent it to sequence, but the result was not correct. I tried to ligate BS-yxjA with BBa_K156032 and ChiA with secretion tag and BBa_J23114 with B0034 in pSB1C3 again, after transformation and colony PCR, I sent them to sequence, BS-yxjA with BBa_K156032 was correct but ChiA with tag was still not correct. Next, I tried to amplify ChiB with NheI restriction sites by using correct template, and I got the correct band this time. I then I picked up colony from hybrid promoter 2-5 plate again, and PCR amplify the inserted part and cultured the correct bands(Fig.17).After I sent the PCR products to sequence, some of them (P3+E1010 and P5+E1010) were still not correct. Thus, I tried to pick up colonies from the original plates again, and in this time, P5+E1010 was correct. Then I tried to transform BBa_J23106 plus mRFP-P0440 again, and also tried to transform BBa_K215002 from 2016 kits, then I picked up colony from that two plates and check BBa_J23106 plus mRFP-P0440 by PCR. After culture and plasmid purification(166.4 ng/μL), I sent it to sequence but it failed again. Also, I picked up colony from VB12-C0012+B0015 plate again, and got correct sequencing result this time. Last, I tried to ligate ChiA with BBa_K125001 and BBa_K125002 again, but I only digested each part with NheI 1 hour to prevent “star activity”. And I tried to ligate them again.
Week 14 (20st-27th September, 2016)
I transformed ChiA plus BBa_K125001 and BBa_K125002, P1+mRFP-P0440 and P4+mRFP-P0440, after I cultured and picked up colony from these plates, and checked the inserted part by PCR. Then I sent them to sequence, but the results were all not correct. Next, I digested BBa_J23106 with EcoRI and SpeI and BS-yxjA+BBa_K156032, YF1+FixJ+B0015 with XbaI and PstI. Then I ligated BBa_J23106 with BS-yxjA+BBa_K156032 and YF1+FixJ+B0015 in pSB1C3. After transformation, I picked up colony from these plates and used PCR to amplify the inserted part. Then I sent them to sequence and only BBa_J23016 with YF1-FixJ+B0015 was correct. BBa_J23016+BS-yxjA-BBa_K516032 sequencing failed at this time. For the last two part of hybrid promoter P3 and P3 with E1010, I tried to use the PCR products from the first time colony PCR, pro3-E1010(4), pro5-E1010(2) (92.1 ng/μL, 89.4 ng/μL) and sent them to sequence, and the results were correct in this time. Then, I tried to co-transformed this light sensor generator (BBa_K19103034) with hybrid promoter 2 and 4, because I have not gotten the sequencing results of P3 and P5. First, I made pSB4K5 vector and I changed the backbone from pSB1C3 to low copy number vector pSB4K5. Then, I did co-transformation of P2+E1010 and P4+E101 with BBa_J23016-YF1-FixJ+B0015 and picked up colony from the plates, then I culture them under dark condition over 24h with control (only contains hybrid promoter with mRFP) and tested fluorescence value and absorbance OD600(Fig. 18). Last, I tried to amplify riboswitch BS-xpt with BBa_K156032 again and sent it to sequence, but the results were still not correct.
Week 15 (28th September-4th October, 2016)
Firstly, I tried to use PCR to rebuild secretion tag prtB by adding a short linker on the C terminal of secretion tag and N terminal of Chitinase A and B. Also, I added a His tag on the C terminal of Chitinase A and B by PCR (annealing temperature are secretion tag:52℃, ChiA+His: 67℃, ChiB+His: 67℃). Then, I did gel purification for purifying these two part and digestion them with BamHI overnight. And I ligate them for 2.5h, and tried to use PCR amplify whole Chitinase A and B with secretion tag part. But only ChiA-His tag+secretion tag succeeded (Fig.19). After I sent ChiA+secretion tag to sequence, the sequencing result was not correct. Then I tried to use gradient PCR to amplify ChiB with secretion tag again (From 48℃-60℃), but it failed again. Next, I sent BBa_J23016+BS-yxjA-BBa_K516032 PCR product to sequence again, and I got the correct sequencing result this time. Then, I culture BBa_R0010 and BBa_R0040 and purified plasmid (60.5 ng/μL, 54.5 ng/μL). After digestion of BBa_R0010 and BBa_R0040 and VB12+C0012-B0015 and VB12+C0040-B0015, and ligated them in PSB1A3. After transformation and colony PCR and sent the correct PRC product to sequence, but only BBa_R0040- VB12+C0012-B0015 had correct sequencing result. Also, I tried to ligate P4+mRFP-P0440 and P1+ mRFP-P0412 and BBa_J23114 with B0034 in pSB1T3. After transformation and colony PCR, I sent them and a previous PCR product of BBa_J23114 with B0034 to sequence. But only the previous PCR product of BBa_J23114 was correct.
Week 16 (5th-10th October, 2016)
Firstly. We ordered original pDawn and pDusk system, and tested them with light and dark condition at the same time and cultured them over 17h. The results showed that pDwan system worked well, but pDusk did work (Fig.20). And then I tested fluorescence value (mcherry 575nm, 601nm) of pDawn and pDusk samples with three replicates. Also, we co-transformed BBa_K592020 (Blue fluorescence protein controlled by original Fixk2) with light sensor generator BBa_K1913034, but under 12h dark condition, we got blue colony which suggested that Fixk2 promoter (BBa_K546000,) did not work well(Fig.21). last, I co-transformed each hybrid promoter plus E1010 part with light sensor generator BBa_K1913034, and cultured them under dark condition over 24h with controls. And I tested fluorescence value and absorbance OD600 with three replicates. The results show on Fig.22.
