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Revision as of 03:26, 19 September 2015

Peking iGEM 2015

Parts

Each part give 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

Favorite Name Type Description Design
 √ BBa_K1689000 Composite sgRNA generator ZHAO Xuejin
 √ BBa_K1689008 Composite Coding sequence of dCas9-Nluc ZHANG Yihao
 √ BBa_K1689009 Composite Coding sequence Cluc-dCas9 ZHANG Yihao
  BBa_K1689010 Compostie Coding sequence of N-luc-dCas9 ZHANG Yihao
  BBa_K1689001 Coding Coding sequence of STV-N-luc ZHANG Yihao
  BBa_K1689002 Coding Coding sequence of Cluc-STV ZHANG Yihao
  BBa_K1689003 Coding Coding sequence of Nluc416-FRB Xiang Li
  BBa_K1689004 Coding Coding sequence of Nluc398-FRB Xiang Li
  BBa_K1689005 Coding Coding sequence of FKBP-Cluc398 Xiang Li
  BBa_K1689006 Coding Coding sequence of FKBP-Cluc394 Xiang Li
  BBa_K1689007 Composite Coding sequence of dCas9-C-luc ZHANG Yihao
  BBa_K1689011 Composite delta alpha segment of lacz fused with dCas9, His tag allows for protein purification Jiaofeng Li
  BBa_K1689012 Composite N terminal of β-lactamase fused with dCas9 Jiaofeng Li
  BBa_K1689013 Compostie NFluc of lactamase fused with dCas9, His tag allows for protein purification Jiaofeng Li
  BBa_K1689014 Coding dCas9 fused with C terminal of beta-lactamase Wei Weijia
  BBa_K1689015 Coding segment F[1,2] of DHFR-dCas9 fusion protein Wei Weijia
  BBa_K1689016 Coding fusion protein of dCas9-F[1,2] Wei Weijia
  BBa_K1689017 Coding DHFR F3 fragment-dCas9 fusion protein Wei Weijia
  BBa_K1689018 Coding dCas9 fused with F3 Wei Weijia
  BBa_K1689019 Coding Δα segment of β-galactosidase fused with dCas9 Wei Weijia
  BBa_K1689020 Coding dCas9 fused with delta omega segment of β-galactosidase Wei Weijia

Parts for paired dCas9 reporter system

Nluc416-FRB (BBa_K1689003), FKBP-Cluc398 (BBa_K1689005), Nluc398-FRB (BBa_K1689004) and 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) and 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) and 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 explore 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.