Difference between revisions of "Team:Wageningen UR/Notebook/toggleswitch"

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  <h2><b>Week 4 (11th-16th July, 2016)</b></h2>
 
  <h2><b>Week 4 (11th-16th July, 2016)</b></h2>
 
<p>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)</p>
 
<p>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)</p>
 +
<h2><b>Week 5 (19th-24th July, 2016)</b></h2>
 +
<p>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℃.</p>
 +
<h2><b>Week 6 (26th July -2nd August, 2016)</b></h2>
 +
<p>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.</p>

Revision as of 16:37, 17 October 2016

Wageningen UR iGEM 2016

 

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.