Difference between revisions of "Part:BBa K1689008"

Latest revision as of 15:44, 27 September 2015

dCas9-N-luc

dCas9-N-luc fusion protein ORF

A catalytically dead Cas9 (dCas9), when co-expressed with a guide RNA, forms a DNA recognition complex which can bind any sequence [1]. Firefly luciferase, widely used as a reporter, is split into two fragments, namely N-luc and C-luc [2]. Each fragment by itself is inactive; when two fragments are reassembled, the enzymatic activity of the original protein would be reconstituted, providing easily measurable readout.

Peking iGEM 2015 fused N-luc to C terminus of dCas9 (Figure 1). Guided by sgRNA, it binds to target DNA sequence. Together with another part, dCas9-C-luc (BBa_K1689007):sgRNA complex, our paired dCas9 (PC) reporter system would work to (Figure 2) to convert the sequence-specific information of pathogenic bacteria's genome (in our case, M. tuberculosis) into easily readable bioluminescence signal.

Figure 1. Schematic cartoon of dCas9-N-luc fusion protein.

Figure 2. Working mechanism of the paired dCas9 (PC) reporter system.

We thoroughly optimized the configuration of our PC reporter system (see [http://2015.igem.org/Team:Peking/Design/PC_Reporter Methods]). Provided that the initial binding of dCas9 to DNA depends on the protospacer adjacent motif (PAM, a short 3’ motif adjacent to target sequence), four sets of sgRNA orientation settings were tested (Figure 3a).To find out how split luciferase-dCas9 fusion strategy influences our PC reporter system, we constructed and tested C-luc-dCas9(BBa_K1689009), dCas9-C-luc (BBa_K1689007) fusion protein to respectively pair with dCas9-N-luc (Figure 3b).

a

b

Figure 3. Thorough optimization on the configuration of our PC reporter system. (a) 4 different sgRNA orientation settings. In orientation PAM-out, the pair of PAM sequences are distal from the spacer sequence, with the 5' end of the sgRNA adjacent to the spacer; in orientation PAM-in, the pair of PAM sequences are adjacent to the spacer sequence, with the 3' end of the sgRNA in proximity to the spacer; in orientation PAM-direct 1 and PAM-direct 2, one PAM sequence is adjacent to and another distal from the spacer. (b) Test on dCas9-N-luc fusion strategies paired with C-luc-dCas9 and dCas9-C-luc across four different sgRNA orientations.

References

1. Lei S. Qi, Matthew H. Larson, Luke A. Gilbert et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152: 1173-1183.

2. Kathryn E. Luker, Matthew C. P. Smith, et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS, 2004, 101: 12288-12293.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1176
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 3
Illegal BamHI site found at 3455
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1689008 short</partinfo>
-dCas9-Nluc fusion protein ORF
+dCas9-N-luc fusion protein ORF
-A catalytically dead Cas9 (dCas9), when coexpressed with a guide RNA, generates a DNA recognition complex which can binds to any targeted gene [1]. Firefly luciferase, widely used as a reporter, is split into two fragments, namely Nluc and Cluc [2]. Each protein fragment by itself is inactive, when two fragments are reassembled, the enzymatic activity of the original protein would be reconstituted, providing easily measurable read out.
+A catalytically dead Cas9 (dCas9), when co-expressed with a guide RNA, forms a DNA recognition complex which can bind any sequence [1]. Firefly luciferase, widely used as a reporter, is split into two fragments, namely N-luc and C-luc [2]. Each fragment by itself is inactive; when two fragments are reassembled, the enzymatic activity of the original protein would be reconstituted, providing easily measurable readout.
-Peking iGEM fused Nluc to C terminus of dCas9 (Figure 1). Guided by sgRNA, it binds to target DNA sequence. Together with dCas9-Cluc [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689007  (BBa_K1689007)]:sgRNA complex, our paired dCas9 (PC) reporter system was constructed (Figure 2) which converts invisible sequence information into measurable bioluminescence.
+Peking iGEM 2015 fused N-luc to C terminus of dCas9 (Figure 1). Guided by sgRNA, it binds to target DNA sequence. Together with another part, dCas9-C-luc [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689007  (BBa_K1689007)]:sgRNA complex, our paired dCas9 (PC) reporter system  would work to (Figure 2) to convert the sequence-specific information of pathogenic bacteria's genome (in our case, <I>M. tuberculosis</I>) into easily readable bioluminescence signal.
 [[File:Peking-dCas9-Nluc.png|400px|]]
-'''Figure 1. Configuration of dCas9-Nluc fusion protein.'''
+'''Figure 1.  Schematic cartoon of dCas9-N-luc fusion protein.'''
 [[File:Peking-CRISPR-Figure2.png|700px|]]
-'''Figure 2. Schematic of the paired dCas9 (PC) reporter system.'''
+'''Figure 2. Working mechanism of the paired dCas9 (PC) reporter system.'''
-We invented a new protocol for testing our PC reporter system (see Methods). Provided that initial binding of dCas9 depends on the protospacer adjacent motif (PAM, a short 3’ motif adjacent to target sequence), four sets of sgRNA orientation settings were also tested (Figure 3a). To find out how split luciferase-dCas9 fusion architecture influences our PC reporter system, we constructed and tested Cluc-dCas9[https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689009 (BBa_K1689009)], dCas9-Cluc [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689007 (BBa_K1689007) ]fusion protein to respectively pair with dCas9-Nluc (Figure 3b).
+We thoroughly optimized the configuration of our PC reporter system (see [http://2015.igem.org/Team:Peking/Design/PC_Reporter Methods]). Provided that the initial binding of dCas9 to DNA depends on the protospacer adjacent motif (PAM, a short 3’ motif adjacent to target sequence), four sets of sgRNA orientation settings were tested (Figure 3a).To find out how split luciferase-dCas9 fusion strategy influences our PC reporter system, we constructed and tested C-luc-dCas9[https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689009 (BBa_K1689009)], dCas9-C-luc [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1689007 (BBa_K1689007) ]fusion protein to respectively pair with dCas9-N-luc (Figure 3b).
 a
@@ Line 28: / Line 28: @@
 [[File:Peking-11.png|700px|]]
-Figure 3. Test on dCas9-Nluc fusion protein across four different sgRNA orientations. (a) 4 different sgRNA orientation settings. In orientation PAM-out, the pair of PAM sequences are distal from the spacer sequence, with the 5' end of the sgRNA adjacent to the spacer; in orientation PAM-in, the pair of PAM sequences are adjacent to the spacer sequence, with the 3' end of the sgRNA in proximity to the spacer; in orientation PAM-direct 1 and PAM-direct 2, one PAM sequence is adjacent to and another distal from the spacer. (b) Test on dCas9-Nluc fusion protein paired with Nluc-dCas9 and dCas9-Nluc across four different sgRNA orientations.
+Figure 3. Thorough optimization on the configuration of our PC reporter system. (a) 4 different sgRNA orientation settings. In orientation PAM-out, the pair of PAM sequences are distal from the spacer sequence, with the 5' end of the sgRNA adjacent to the spacer; in orientation PAM-in, the pair of PAM sequences are adjacent to the spacer sequence, with the 3' end of the sgRNA in proximity to the spacer; in orientation PAM-direct 1 and PAM-direct 2, one PAM sequence is adjacent to and another distal from the spacer. (b) Test on dCas9-N-luc fusion strategies  paired with C-luc-dCas9 and dCas9-C-luc across four different sgRNA orientations.
@@ Line 34: / Line 34: @@
 ==References==
 .	Lei S. Qi, Matthew H. Larson, Luke A. Gilbert et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 2013, 152: 1173-1183.
 .	Kathryn E. Luker, Matthew C. P. Smith, et al. Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS, 2004, 101: 12288-12293.