Difference between revisions of "Part:BBa K2965021"
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===Usage and Biology=== | ===Usage and Biology=== | ||
+ | AsCas12a RNPs bind to DNA and start to move along it by a hopping mechanism. When the AsCas12a RNPs encounter a potential target site containing a PAM sequence, they begin to initiate R-loop formation by forming base-pair hybrids between the crRNA and the target DNA strand. If sufficient matched nucleotides exist in the PAM-proximal region, the R-loop formation will be stable. After the stable R-loop conformation is formed, AsCas12a, via the activity of its RuvC domain, first cleaves the non-target DNA strand and then cleaves the opposite, target DNA strand, regardless of the presence of a PAM sequence. Finally, AsCas12a rapidly releases the PAM-distal DNA fragment but continues to hold the PAM-proximal portion of DNA[1]. | ||
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+ | ===Characterization=== | ||
+ | ====Result==== | ||
+ | We link AsCas12a fragment and pET-28a(+) plasmid to AbCas12a expression plasmid with His tag. Then we transfer recombinant plasmid into E. coli BL21 and test it by colony PCR. Result is shown in Figure 1 below. We also verify it by TSINGKE Biologocal Technology Institute sequencing. | ||
+ | [[File:BBa K2965021-Figure 1. AsCas12a colony PCR..png|center|500px|thumb|''' Figure 1. AsCas12a colony PCR.''']] | ||
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+ | We induce AsCas12a expression and purify it by gravity-flow column with Ni-NTA Sefinose(TM) Resin. SDS-PAGE and Western Blot is used to test protein purification as shown in Figure 2 and Figure 3. | ||
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Revision as of 07:47, 21 October 2019
AsCas12a (AsCpf1)
AsCas12a (AsCpf1) comes from Acidaminococcus sp. BV3L6, it is a programmable DNA endonuclease guided by a single CRISPR RNA (crRNA). Targeting requires a crRNA complementary to the target site as well as a 5' TTTN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Once it recognizes its target, it will be activated and exhibits a “collateral effects” of promiscuous DNase activity.
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 196
Illegal PstI site found at 1258
Illegal PstI site found at 3667
Illegal PstI site found at 3865 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 196
Illegal PstI site found at 1258
Illegal PstI site found at 3667
Illegal PstI site found at 3865 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 380
Illegal BglII site found at 1922
Illegal BglII site found at 2231 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 196
Illegal PstI site found at 1258
Illegal PstI site found at 3667
Illegal PstI site found at 3865 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 196
Illegal PstI site found at 1258
Illegal PstI site found at 3667
Illegal PstI site found at 3865
Illegal NgoMIV site found at 3466 - 1000COMPATIBLE WITH RFC[1000]