Part:BBa_K3028000
N-terminal part of beta-lactamase fused with dCas9 and double terminator
N-terminal part of beta-lactamase fused with dCas9 and double terminator
Part BBa_K1689013 is an N-terminal fragment of β-lactamase fused with dCas9. dCas9 enzyme is also known as a catalytically dead Cas9 enzyme[1]. It lacks traditional endonuclease activity (it does not cleave DNA) but preserves its ability to bind targets very specifically. This part was modified by IGEM_Peking team for DNA detection purposes. β-Lactamases - bacterial enzymes that can hydrolyze the amide bond of the β-lactam ring, hence inactivate β-lactam antibiotics (AmpR). Split β-Lactamase (196|198) [2] is used as a reporter, for example, to study different protein-protein interactions. When fused to dCas9, it can report the presence of a specific target by regaining its enzymatic activity.
Our part is a composition of the most commonly used strong double terminator and BBa_K1689013. It was necessary to make such a composite part, because of the side effects of Cas synthesis for cells. It was shown that even in the absence of gRNA, Cas9 and dCas9 expression hindered cell growth.
[1] Repurposing CRISPR As An RNA-Guided Platform For Sequence-Specific Control Of Gene Expression. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., and Lim, W. A. 2013, Cell Vol. 152, pp. 1173-1183.
[2] Beta-lactamase complementation assays as in vivo and in vitro sensors of protein–protein interactions Galarneau A, Primeau M, Trudeau L, Michnick S; Nature Biotechnology; 2002 vol: 20 (6) pp: 619-622
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1693
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4
Illegal BamHI site found at 3972 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |