Difference between revisions of "Part:BBa K4297077"

Revision as of 06:57, 11 October 2022

PL-CBE

The composite part contains Lambda cI , cytidine deaminase, Uracil DNA glycosylase inhibitor and LVA degradation tag. CDA catalyzes the deamination of cytosine in the top (non-complementary) strand located within 15-20 bases upstream of PAM, resulting in a C-T mutation.

Fig. The plasmid construction process

Usage and Biology

It has been shown that extending the incubation time of the strain will increase the editing efficiency, which often leads to over-editing. To overcome this problem, we replaced the constitutive promoter PCas in BBa_K3868097 with the inducible promoter Plambda cI. Further, we transformed optimized CBE and sgRNA (targeting the lacZ gene) into BL21(DE3) for base editing test, which were induced at 42°C for 0h,6h, 12h, 18h, and 24h, respectively. The experimental results showed that the number of white spots was found to be positively correlated with the induction time (Fig 3A). After calculations, the editing efficiency was 8.6% (3/35), 30.7% (23/75), 78.2% (43/55), 86.7% (60/69), and 96.0% (69/72), respectively (Fig 3B). To ensure the authenticity of the experiment, we selected five white spots in each case for sequencing, and all were found to have introduced nonsense mutations in lacZ.

Fig. (A) The test workflow for CBE editorial moderation. (B) The editing efficiency under different induction times at 42℃.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2097
Illegal NheI site found at 6019
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 5432
Illegal BamHI site found at 4376
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 5111
Illegal AgeI site found at 5757
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 988
Illegal BsaI site found at 5114
Illegal BsaI site found at 5760
Illegal BsaI.rc site found at 5108
Illegal BsaI.rc site found at 5754
Illegal BsaI.rc site found at 6028

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 ===Usage and Biology===
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+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;It has been shown that extending the incubation time of the strain will increase the editing efficiency, which often leads to over-editing. To overcome this problem, we replaced the constitutive promoter PCas in BBa_K3868097 with the inducible promoter Plambda cI. Further, we transformed optimized CBE and sgRNA (targeting the lacZ gene) into BL21(DE3) for base editing test, which were induced at 42°C for 0h,6h, 12h, 18h, and 24h, respectively. The experimental results showed that the number of white spots was found to be positively correlated with the induction time (Fig 3A). After calculations, the editing efficiency was 8.6% (3/35), 30.7% (23/75), 78.2% (43/55), 86.7% (60/69), and 96.0% (69/72), respectively (Fig 3B). To ensure the authenticity of the experiment, we selected five white spots in each case for sequencing, and all were found to have introduced nonsense mutations in lacZ.
-  It has been shown that extending the incubation time of the strain will increase the editing efficiency, which often leads to over-editing. To overcome this problem, we replaced the constitutive promoter PCas in BBa_K3868097 with the inducible promoter Plambda cI. Further, we transformed optimized CBE and sgRNA (targeting the lacZ gene) into BL21(DE3) for base editing test, which were induced at 42°C for 0h,6h, 12h, 18h, and 24h, respectively. The experimental results showed that the number of white spots was found to be positively correlated with the induction time (Fig 3A). After calculations, the editing efficiency was 8.6% (3/35), 30.7% (23/75), 78.2% (43/55), 86.7% (60/69), and 96.0% (69/72), respectively (Fig 3B). To ensure the authenticity of the experiment, we selected five white spots in each case for sequencing, and all were found to have introduced nonsense mutations in lacZ.
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