Difference between revisions of "Part:BBa K2927051"

Revision as of 07:28, 16 October 2019

pHMT-LbCas12a plasmid (LbCas12a protein purification)

To express and purify Cas12a protein, we combine T7 promoter, poly Histidine-tag (10XHis), Maltose binding protein (MBP), and TEV recognizing motif with Cas12 coding sequences. T7 promoter can produce high yields of protein in E. coli with T7 RNA polymerase expression. 10XHis and MBP are used for protein purification through their high affinity to Nickel (II) ion and maltose, respectively. TEV recognizing motif separate 10XHIS and MBP from Cas12a, which is used to elute untagged Cas12a protein after purification. The composition 10X His-MBP-TEV site-Cas12a is called pHMT-Cas12a.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 4921
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 453
Illegal BglII site found at 2600
Illegal BglII site found at 3337
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 4251
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 151
Illegal BsaI.rc site found at 2664

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 <partinfo>BBa_K2927051 short</partinfo>
-T7 promoter can produce high yields in the transcription of E. coli when T7 RNA polymerase is present. The sequence we designed is for Cas12a protein expression and purification. Therefore, E.coli BL21 was used for producing our Cas12a protein. 10 times histidine and MBP are used for protein purification, and the TEV cutting site lets Cas12a protein separate from the composition (10X His-MBP-TEV site-Cas12a). 10 times histidine has a strong affinity with nickel ion, and the nickel ion is bind with nickel column. Therefore, we can separate the composition from the solution by nickel ion after breaking bacteria. In the second step of protein purification, the TEV enzyme cut the TEV cutting site for separate Cas12a protein from the composition. Next, we will get Cas12a protein from solution after gathering 10X His- MBP on nickel column.
+To express and purify Cas12a protein, we combine T7 promoter, poly Histidine-tag (10XHis), Maltose binding protein (MBP), and TEV recognizing motif with Cas12 coding sequences. T7 promoter can produce high yields of protein in E. coli with T7 RNA polymerase expression. 10XHis and MBP are used for protein purification through their high affinity to Nickel (II) ion and maltose, respectively.  TEV recognizing motif separate 10XHIS and MBP from Cas12a, which is used to elute untagged Cas12a protein after purification. The composition 10X His-MBP-TEV site-Cas12a is called pHMT-Cas12a.
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