Difference between revisions of "Part:BBa K3868098"
Line 32: Line 32:
 
<div align="center">
 
<div align="center">
 
     <figure>
 
     <figure>
−
         <img src="https://2021.igem.org/wiki/images/3/3b/T--NNU-China--part-engineering_success-1.png" width="40%" style="float:center">
+
         <img src="https://2021.igem.org/wiki/images/3/3b/T--NNU-China--part-engineering_success-1.png" width="30%" style="float:center">
 
         <figcaption>
 
         <figcaption>
 
         <p style="font-size:1rem">
 
         <p style="font-size:1rem">

Revision as of 09:20, 19 October 2021


pTargetS

pTargetS plasmid contains pj23119, sgRNA-1 and sgRNA-2 etc.Two different sgRNA expression frames were tandemly linked, allowing GGGGGGGG to be covered, resulting in a more diverse editing outcome.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 182
    Illegal EcoRI site found at 552
    Illegal XbaI site found at 188
    Illegal XbaI site found at 558
    Illegal SpeI site found at 73
    Illegal SpeI site found at 443
    Illegal PstI site found at 200
    Illegal PstI site found at 570
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 182
    Illegal EcoRI site found at 552
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 50
    Illegal NheI site found at 377
    Illegal NheI site found at 400
    Illegal NheI site found at 420
    Illegal SpeI site found at 73
    Illegal SpeI site found at 443
    Illegal PstI site found at 200
    Illegal PstI site found at 570
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 182
    Illegal EcoRI site found at 552
    Illegal BglII site found at 212
    Illegal BglII site found at 582
    Illegal XhoI site found at 236
    Illegal XhoI site found at 606
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 182
    Illegal EcoRI site found at 552
    Illegal XbaI site found at 188
    Illegal XbaI site found at 558
    Illegal SpeI site found at 73
    Illegal SpeI site found at 443
    Illegal PstI site found at 200
    Illegal PstI site found at 570
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 182
    Illegal EcoRI site found at 552
    Illegal XbaI site found at 188
    Illegal XbaI site found at 558
    Illegal SpeI site found at 73
    Illegal SpeI site found at 443
    Illegal PstI site found at 200
    Illegal PstI site found at 570
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

        The dual plasmids system of CBE was designed, built and tested. The pCBE plasmid contains the lambda operator, cytidine deaminase, Uracil DNA glycosylase inhibitor and LVA degradation labels. Based on the pCas, the pCBE (BBa_K3868097) was successfully constructed (Fig. 3A). To extend the editing range, two different sgRNA expression frames were tandemly linked, allowing GGGGGGGG to be covered, resulting in a more diverse editing outcome. Based on the pTarget, the pTargetS plasmid (BBa_K3868098) was successfully constructed (Fig. 3B), and the sequences of sgRNA1 and sgRNA2 was showed in Fig. 3C. The CBE / sgRNA complex can bind to the double-stranded DNA to form an R-loop in a sgRNA and PAM-dependent manner. CDA catalyzes the deamination of cytosines located at the top (non-complementary) strand within 15–20 bases upstream from PAM, which results in C-to-T mutagenesis.

Fig 3. A and B. The dual plasmid system was designed and used for CBE system. C. A schematic model for CBE.

Results

         During construction of the library, 90 single colonies were randomly selected for sequencing. It was found that 48 variants with different RBS sequence were identified from 90 samples, with an editing efficiency of only 53%. However, it is noteworthy that the transformants were grown on the solid medium for longer time, the reproducibility of the results gradually increased, and majority of variant RBS sequences became GAAAAAAG (Fig.4), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.

Fig 4. Schematic representation of the changes in G and A abundance of the RBS variant sequences of T7 RNAP obtained from CBEs experiments.

Reference

1. Gong G, Zhang Y, Wang Z, Liu L, Shi S, Siewers V, Yuan Q, Nielsen J, Zhang X, Liu Z. GTR 2.0: GRNA-tRNA array and Cas9-ng based genome disruption and single-nucleotide conversion in Saccharomyces cerevisiae. ACS synthetic biology. 2021; 10: 1328–1337.

2. Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X. Glycosylase base editors enable C-to-A and C-to-G base changes. Nature Biotechnology. 2021; 39: 35–40.