Difference between revisions of "Part:BBa K1875006"

Revision as of 05:06, 19 October 2016

A 20 bp target sequence and PAM to make guide operator 8

The [http://2016.igem.org/Team:BostonU BostonU 2016] iGEM team synthesized guide operator sequences composed of a 20 base pair target sequence and a PAM sequence. The target sequence was made using a random DNA generator and entered into the [http://crispr.mit.edu/ CRISPR Design Optimization] tool for the human genome (hg19). It was chosen among approximately 1000 other randomly generated target sequences due to its high optimization score ( > 98%) and high expression in an initial screen containing iRFP (figure 1).

The target sequence is expressed by a guide RNA (gRNA) expression vector (BBa_K1875011) from the BostonU Gemini Library. The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter (BBa_K1875014) to activate a downstream gene of interest (figure 2).

Figure 1: Initial screen of BostonU's first 18 operator reporters containing GFP

Figure 2: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1875006 short</partinfo>
-This guide is expressed by BBa_K1875012 and is the target sequence on BBa_K1875015
+The [http://2016.igem.org/Team:BostonU BostonU 2016] iGEM team synthesized guide operator sequences composed of a 20 base pair target sequence and a PAM sequence.  The target sequence was made using a random DNA generator and entered into the [http://crispr.mit.edu/ CRISPR Design Optimization] tool for the human genome (hg19).  It was chosen among approximately 1000 other randomly generated target sequences due to its high optimization score ( > 98%) and high expression in an initial screen containing iRFP (figure 1).
+The target sequence is expressed by a guide RNA (gRNA) expression vector ([https://parts.igem.org/Part:BBa_K1875011 BBa_K1875011]) from the BostonU Gemini Library.  The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter ([https://parts.igem.org/Part:BBa_K1875014 BBa_K1875014]) to activate a downstream gene of interest (figure 2).
+[[File:T--BostonU--iRFP_screen.png|400px|thumb|left|Figure 1: Initial screen of BostonU's first 18 operator reporters containing GFP]][[File:T--BostonU--ProjectDescription.png|400px|thumb|center|Figure 2: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description]]
 <!-- Add more about the biology of this part here