Team:Peking R/Notebook/MT

I participated in designing of the project and constructing the characterization system, focusing on the regulatory elements utilized in the project

MU Tong’s Notebook

7.1~7.7
Preparation of biobrick parts that would be used in our project from the 2011 distribution, including transformation and mini-prep, followed by restriction digestion with certain enzymes according to the standard assembly assay.
Designing and synthesizing the primers and a regulatory element.

7.8
Experimental trouble shooting: testing and optimizing the protocols of double digestion, PCR and ligation.

7.9~7.11
Constructing the theophylline hammerhead regulatory system, including constructing hammerhead element with OE PCR, double digestion and ligation following the standard assembly assays.
Sequence correct. J

7.12~7.17
Constructing the RBS strength characterization system with random primers using PCR. Optimizing the protocol and preliminary characterization of the RBS strength.
However, the results suggest that the system is polluted by template.
Designing new primers.

7.18~7.25
Constructing the RBS characterization system by another approach.
Sequence correct.
Preliminary characterization of the system.

7.26~8.5
Assembly of the Theophylline responsive hammerhead ribozyme (theo HH) with the Cd Intron utilizing two approaches, Standard Assembly and Gibson Assembly. Further attaching this regulatory element to a GFP reporter (pBAD+RBS+GFP+Terminator) to demonstrate the responsive action to theophyline.

8.6~8.14
Constructing the Parental T4td Intron and assembling it into the GFP reporter. Fusing the parental intron with a theophylline responsive aptamer.
Altering the recognization specificity by changing the theophylline aptamer into a Thiamine Pyrophosphate (TPP) responsive hammerhead ribozyme (TPP HH).
Both elements show limited responsive to the ligands.

8.30~9.7
Designing and synthesizing the primers of AND gates.
Constructing the AND gates with theophylline riboswitches residing downstream the pSal promoter.

9.8~9.20
Testing and optimizing the protocols of characterizing the ligand responsive AND gates.
Preliminary characterization of the AND gates.
Characterization of specific AND gates that performed obvious ligand responsive actions in the preliminary characterization. Collecting the data for modeling.

9.21~9.28
Preparing for the part submission: collecting well characterized and standardized parts from team members, documenting parts in the Part Registry, submitting the parts.
Further characterization of the AND gates.

9.29~10.4
Collecting experimental data and protocols.
Writing manuscript for wiki.