Difference between revisions of "Part:BBa K2017008"

Revision as of 15:23, 22 October 2016

35s + Ga20ox consense + RSIAT-Luciferase + Tnos

Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3').

It includes a promoter 35s, 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT linker (BBa_K2017004) and the terminator Tnos.

Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 871
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 66
Illegal BsaI site found at 2544
Illegal BsaI.rc site found at 2831
Illegal SapI.rc site found at 1690

Usage and Biology

The gRNA testing system is a modular and standard Phytobrick device.

Figure 1: Schematic of the function of the genetic circuit that composes the gRNA Testing System. Only when DSB and subsequent repair is produced the luciferase’s open reading frame will shift to the correct one (ON state).

Figure 2: Comparison of the different mutation ratios of RSIAT linker version. Luminescence ratios from luciferase constitutive expression regulated by pNOS and 35s promoters are also shown. As can be seen luciferase ratios from Ga20KO-NTgRNA are higher than constitutively expressed luciferase.

Figure 3:Comparison of the different mutation ratios of RSIAT linker version. As can be seen there is no statistical difference between Ga20PCR-Ga20gRNA and its negative control Ga20PCR-NTgRNA. However Ga20cons-Ga20gRNA ratio is significantly higher than its negative control Ga20cons-NTgRNA

Revision as of 15:21, 22 October 2016 (view source) Movigusa (Talk \| contribs) ← Older edit		Revision as of 15:23, 22 October 2016 (view source) Movigusa (Talk \| contribs) Newer edit →
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	The gRNA testing system is a modular and standard Phytobrick device.		The gRNA testing system is a modular and standard Phytobrick device.

−	[[Image:~~Gts_target_2_upv~~.png\|600px\|thumb\|center\|'''Figure 1:''' Schematic of the function of the genetic circuit that composes the gRNA Testing System. Only when DSB and subsequent repair is produced the luciferase’s open reading frame will shift to the correct one (ON state).]]	+	[[Image:Gtstargetdesign.png\|600px\|thumb\|center\|'''Figure 1:''' Schematic of the function of the genetic circuit that composes the gRNA Testing System. Only when DSB and subsequent repair is produced the luciferase’s open reading frame will shift to the correct one (ON state).]]