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Revision as of 06:54, 16 November 2015

Peking iGEM 2015

Parts

Each part gives us a new experience.

Favorite Parts

Nluc-dCas9 fusion protein
(BBa_K1689010)

Nluc was fused to the N terminus of dCas9 to form the Nluc-dCas9 fusion protein. It is used to bind a DNA sequence guided by sgRNA. Paired with Cluc-dCas9 fusion protein (BBa_K1689009) and sgRNAs, our paired dCas9 reporter system were constructed.

Cluc-dCas9 fusion protein
(BBa_K1689009)

Cluc was fused to the N terminus of dCas9 to form the Cluc-dCas9 fusion protein. It is used to bind a DNA sequence guided by sgRNA. Paired with Nluc-dCas9 fusion protein (BBa_K1689010) and sgRNAs, our paired dCas9 reporter system were constructed.

sgRNA generator
(BBa_K1689000)

sgRNA generator is used for generating sgRNA which recognizes targeted DNA, then guiding dCas9 to it.

Part List

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Parts for paired dCas9 reporter system

Nluc416-FRB (BBa_K1689003),
FKBP-Cluc398 (BBa_K1689005),
Nluc398-FRB (BBa_K1689004),
FKBP-Cluc394 (BBa_K1689006)
are used to validate the reconsitituation activity of split luciferase.

Nluc-dCas9(BBa_K1689010),
Cluc-dCas9(BBa_K1689009),
dCas9-Nluc (BBa_K1689008),
dCas9-Cluc (BBa_K1689007)
fusion proteins are used for constructing paired dCas9 reporter system.

Δα-dCas9 (BBa_K1689011),
dCas9-Nlact (BBa_K1689012),
Nlact-dCas9 (BBa_K1689013),
dCas9-Clact (BBa_K1689014),
F[1,2]-dCas9 (BBa_K1689015),
dCas9-F[1,2] (BBa_K1689016),
F3-dCas9 (BBa_K1689017),
dCas9-F3 (BBa_K1689018),
dCas9-Δα (BBa_K1689019),
Δω-dCas9 (BBa_K1689020)
are used for further development of dectection.

Parts for Molecular Beacon

STV-Nluc (BBa_K1689001),
Cluc-STV (BBa_K1689002)
are used for binding to molecular beacon.

Parts for sgRNA generation

sgRNA generator (BBa_K1689000) is used for generating sgRNA

Part Collection

2015 Peking iGEM has not only transformed the sequence information into detectable bioluminescence, but also explored a wide range in which Paired dCas9 (PC) Reporter System is able to be applied. Since we have chosen split enzyme as our reporter, it can be substituted by various kinds of enzymes, thus the form of read-out will be abundant at the same time. That is, our PC reporter system has successfully provided a platform to produce a variety of signals.

We combine the specific sequence binding activity of dCas9 with diverse characteristics of split enzymes, thus creating a part collection named "PC Reporters Collection".

The Collection includes:

dCas9-split luciferase Parts

Nluc-dCas9 (BBa_K1689010)
Cluc-dCas9 (BBa_K1689009)
dCas9-Nluc (BBa_K1689008)
dCas9-Cluc (BBa_K1689007)

dCas9-split Dihydrofolate Reductase Parts

F[1,2]-dCas9 (BBa_K1689015)
F3-dCas9 (BBa_K1689017)
dCas9-F[1,2] (BBa_K1689016)
dCas9-F3 (BBa_K1689018)

dCas9-split β–Lactamase Parts

dCas9-Nlact (BBa_K1689012)
Nlact-dCas9 (BBa_K1689013)
dCas9-Clact (BBa_K1689014)

dCas9-splitβ–Galactosidase Parts

Δα-dCas9 (BBa_K1689011)
dCas9-Δα (BBa_K1689019)
Δω-dCas9 (BBa_K1689020)

Among them, the PC reporters with split luciferase oxidizes luciferin and gives out bioluminescence signal, while the other three reporters take electric signal as their output, which is also able to be detected and qualified.