Difference between revisions of "Team:Jilin China/Composite Part"
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Revision as of 11:29, 8 October 2018
COMPOSITE PART
Composite Part
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Editing Alert!
This page is used by the judges to evaluate your team for the medal criterion or award listed below.
Note
This page should list all the composite parts your team has made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page. Remember judges will only look at the first part in the list for the Best Basic Part award, so put your best part first!
Best Basic Part Special Prize
To be eligible for this award, this part must adhere to Registry sample submission guidelines and have been sent to the Registry of Standard Biological Parts. If you have a part you wish to nominate your team for this special prize, make sure you add your part number to your judging form and delete the box at the top of this page.
Please note: Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. -
Overview
This year, we added a total of xx composite parts, including cold-induced RNA thermosensors consisting of Anderson promoter J23104 and cspA mRNA, and a color-selection device designed for Goldengate assembly. We decided to use our composite part B123456 as a strong contender for the Best composite part.
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Color-selection Device for GoldenGate Assembly
We use the color-selection device for goldengate assembly to select the right plasmids based on the color change of monoclonal colonies. The Goldengate assembly is a process of seamless assembly by using IIs-type restriction endonucleases. In our project, we decided to use goldengate assembly to construct our vast number of measurement devices. However, for the successful construction of plasmids, the selection has become a key step in limiting the speed of construction. In order to solve this problem, Jilin_China designed the following construction device.
Our construction device includes a promoter J23101 and chromoprotein tspurple, which can be replaced by the BbsI restriction endonuclease, cjblue that can be replaced by the BsaI restriction endonuclease, conserved sequence and reporter protein sfGFP. Such a composition can be realized that when both the replacement part I and the replacement part II are not replaced, the colony exhibits the color of the tspurple, and when the replacement part I is replaced with another promoter by using BbsI, the colony appears blue, and the unsuccessful colony appears Purple. When BsaI was used to replace Part II, the colony showed the original milky white color of the bacteria, and the blue color colony is unsuccess. The specific color changes are as follows:
Through the visible color changes, we can select successful colonies from the culture dish. In this way, we can eliminate the gel midi purification work, reduce the workload and increase the work efficiency compared with the traditional construction method. There is also a direction in the choice of picking up monoclonal colonies.
After actual experiments, we can successfully build more than 200 plasmids in one day, and the sequencing success rate is over 95%, which is a very exciting result.
Here, we provide a method for efficient and seamless assembly. Users can add more replacements parts using different Type II restriction endonucleases according to their actual conditions, or add a conserved sequence between the replacement parts.
Our strength lies in combining high-efficiency goldengate assembly with chromoprotein selection for greater accuracy and productivity.
If you want to know more about our construction, please visit our construction page, where we will detail the principles of Goldengate.
>Click here to explore more information! < We have recorded the detailed protocol of the goldengate assembly on the protocol page. Other users can refer to our protocol to design their own experiments (the amount of each added reagent in the protocol has been reduced to a minimum, it is not recommended to continue to reduce the amount. At the same time, when actually selecting other types of Type IIs restriction endonucleases, you should test whether the enzyme can work normally in T4 buffer. We have tested T4 ligase and T4 buffer from NEB and Takara to use our system, others has not tested yet.) .
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Cold-induced RNA Thermosensor Parts
In addition, we combined our cold-induced RNA thermosensors (cspA mRNA) with the promoter J23104 as a composite part for uploading, which is more conducive to other users directly, users can also only use a cold-induced RNA thermosensor according to their own needs.