Part:BBa_K1532000

Cla I cutting site (methylation insensitive)

This part is the cutting site of the restriction endonuclease Cla I that is methylation insensitive. The endonuclease Cla I can specifically recognize the sequence of this part and cut the double helix into two cohesive ends. You can use this part when you are trying to add an extra cutting site inside of your system in order to make it open-edited.

Part Description

Usage and Biology

The enzyme Cla I is a restriction endonuclease found in Caryophanon latum. It can specifically recognize the sequence ...ATCGAT... ...TAGCTA... And cut it into ...AT CGAT... ...TAGC TA... At the condition of 37 degrees Celsius. Notably, the methylation Sensitivity results comes out to be that Cla I is sensitive to the restriction methylase Dam for there's a possibility that the base pair right after the ...GAT turns out to be cytosine and trigger the methylation to add a methyl at the N6 site of the adenine (GmATC). This effect may block the cutting process of the Cla I. In this part, we standardized the cutting sequence of the Cla I so that a new cutting site can be added into the system conveniently to leave the system opened for further edit. Also, the sequence we chose made it sure that this site will not be affected by the methylation.

Cutting and Agarose Electrophoresis

Methods

After the assembly ,the plasmid was transferred into the Competent E. coli DH5α(which is dam+). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with Cla I restriction endonuclease bought from TAKARA.

The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Results

The result of the agarose electrophoresis was shown on the following picture. As is shown, the plasmid contains BBa_K1532000 can be cut by the Cla I enzyme under the given circumstances without been affected by the methylation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]