Team:Peking R/Notebook/XQY

I am responsible for the selection of a useful adenine sensing hammerhead ribozyme, which will be used in the regulation of our pathway. I am also responsible for the human practice project.

06.26
1, prepare for our experience, make some solution.
2, transformation of selection-plasmid into DH5α which will be used in the future.

06.27
1, Miniprep of the plasmid

06.28
1,pick one clones and shave at 37℃ to amplify the bacterium. Then dilute it with M9/LB medium.

06.29
1, preparation medium

06.30
1, test the growth curve of HD5α in M9 medium

07.03
1, pick one clones and shave at 37℃ in LB medium for 1h, then culture in M9 medium over night.

07.04
1, test the selection protocol in TOP10 and DH5α

07.05
1,introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 25 μL reaction system (Phusion DNA polymerase)
2, Electrophoresis PCR production in 0.6% agarose gel
3, itroduce wild-type hammerhead ribozyme into selection plasmid by PCR in 25 μL reaction system (Taq DNA polymerase)
4, transformation of selection-plasmid into TOP10
5, Electrophoresis PCR production in 0.6% agarose gel
6, digestion the PCR production with DpnI and purify
7, transformation of the above production into DH5α

07,06
1, optimize phusion PCR system for the amplify of wile-type hammerhead selection plasmid.
2,transformation of wile-type hammerhead selection plasmid into DH5α.
3, Miniprep of the selection plasmid
4, Electrophoresis PCR production in 0.6% agarose gel
5,ligation of 0705 digestion production, then transformation it into DH5α
6, introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 20 μL reaction system (pfu DNA polymerase)

07.07
1， Electrophoresis 0706 PCR production in 0.6% agarose gel
2， introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 20 μL reaction system (PrimeSTAR)
3，transformation of 0706 ligation production

07.08
1, digestion the 0705 PCR production by Klenow
2, purify the above production, then ligation
3, transformation of the above production into DH5α

07．09
1，introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 50 μL reaction system (Taq) purify the production, digestion by DpnI, purify the production, digestion by Klenow, heat inactivation, treat the production by Kinase and ligase
2, transformation of the above production into DH5α
3, pick one clones and shave at 37℃ to amplify the bacterium
4, Miniprep of the wild-type hammerhead ribozyme selection plasmid
5, introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 20μL reaction system (pfu)

07,10
1, Electrophoresis 0709 miniprep production
2, introduce adenine aptamer into wild-type hammerhead ribozyme by PCR in 20μL reaction system (Taq)
3, introduce adenine aptamer into wild-type hammerhead ribozyme by PCR in 20μL reaction system (Taq Mix)
4, introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 50 μL reaction system (MutanBEST kit), Electrophoresis the PCR production

07,11
1, pick one clones and shave at 37℃
2, Miniprep of the wild-type hammerhead ribozyme selection plasmid, Electrophoresis miniprep production
3, introduce adenine aptamer into wild-type hammerhead ribozyme by PCR in 20μL reaction system (Taq Mix), Electrophoresis the PCR production
4, introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 50 μL reaction system (phusion DNA polymerase & MutanBEST kit), purify the production, digestion by DpnI, transformation of the production into DH5α

07.12
1, transformation of the 0711 production into trans1-blue
2, introduce wild-type hammerhead ribozyme into selection plasmid by PCR in 25 μL reaction system (PrimeSTAR) and introduce adenine aptamer into wild-type hammerhead ribozyme by PCR in 20μL reaction system
3，transformation of the PCR production into DH5α
4,try to construct double terminater part

07.13-7.15
1, transformation of AND Gate plasmid (from Haoqian Zhang),B0015 plasmid, B0025 Plasmid into DH5α, Miniprep of these plasmids

07,16-07.26
1, construct adenine hammerhead ribozyme selection plasmid with 12N and transformation it into DH5α
2, trouble shoot and make the selection system work well, optimize the selection system

07.27-08.26
Select adenine up-regulate hammerhead ribozyme

07.26-08.2
Test different expression level of different RBS sequence through plasmid constructed by Mu Tong

08,03-08,14
1,Test the effect of different coding sequence to effectiveness of N8-3 and 1G1 theophylline up-regulate RNA controller through plasmids constructed by Hao Siyang
2, introduce 1G1 theophylline up-regulate RNA controller into AND Gate by Gibson assembling.
3, cotransformate the above plasmid and T7 primer- GFP plasmid into DH5a and test the expression of GFP.
4, construct adenine hammerhead ribozyme selection plasmid with 16N and transformation it into DH2a
5, Test different expression level of different RBS sequence through plasmid constructed by Mu Tong

08,27-09,05
1, introduce 5-methylxanthine respond aptamer into wild-type-hammerhead-ribozyme- selection-plasmid
2, construct always-on and always-off thiamine pyrophosphoric acid respond riboswitch based on selection-plasmid from Yohei Yokobayashi

09,06-09,16
Select 5-methylxanthine up regulate hammerhead ribozyme
Construct describe-plasmid based on selected adenine up-regulate hammerhead ribozyme

09,17-09,20
Test the growth curve of E. coli in M9 medium adding 0.1mg/mL adenine
Do Mock selection through the dual selection system
Describe the effectiveness of selected adenine up-regulate hammerhead ribozyme