Team:Tsinghua/Composite Part
Composite Part
| Number | Name | Short Description | Long Description |
|---|---|---|---|
| [1] | BBa_K2558201 | dCas9 generator with Anderson weak promotor | Part K2558201 is a generator of dCas9, which is a mutant of Cas9 without the DNase activity. dCas9 preserves the ability to bind specific gRNA and then gRNA’s complementary DNA strand. Therefore dCas9/gRNA complex is able to inhibit expression without damaging DNA. As dCas9 is detrimental to the growth of bacteria, we put it after promoter J23114 with B0030 to make sure that dCas9 will be expressed at a constant level with minimal side effect on bacterial growth. |
| [2] | BBa_K2558202 | lacI-regulated, lambda pL hybrid promotor test device | This part contains constantly expressed lacI, and plac-controlled sfGFP as our reporter. We aim to use this construct to test the function of lacI-regulated, lambda pL hybrid promoter by treating the bacteria with different dosages of IPTG and measuring the strength of GFP expression. |
| [3] | BBa_K2558203 | Ptac test device with Anderson strong-expressed lacI | Tac promotor is a hybrid between trp and lacUV5 promoters. Tac promotor is a kind of strong E. coli promotor and is IPTG inducible. This device contains constitutively (BBa_J23100, Anderson strong promotor) expressed lacI protein and Ptac controlled sfGFP reporter. We designed this device along with K2558204 and K2558205, which are the same except for the strength of the constitutive lacI expression. We aim to specify the relation between the level of lacI production and the quality of IPTG induced expression. |
| [4] | BBa_K2558204 | Ptac test device with Anderson medium-expressed lacI | Tac promotor is a hybrid between top and lacUV5 promoters. Tac promotor is a kind of strong E. coli promotor and is IPTG inducible. This device contains constitutively (BBa_J23110, Anderson medium promotor) expressed lacI protein and Ptac controlled sfGFP reporter. We designed this device along with K2558203 and K2558205, which are the same except for the strength of the constitutive lacI expression. We aim to specify the relation between the level of lacI production and the quality of IPTG induced expression. |
| [5] | BBa_K2558205 | Ptac test device with Anderson weak-expressed lacI | Tac promotor is a hybrid between top and lacUV5 promoters. Tac promotor is a kind of strong E. coli promotor and is IPTG inducible. This device contains constitutively (BBa_J23114, Anderson weak promotor) expressed lacI protein and Ptac controlled sfGFP reporter. We designed this device along with K2558203 and K2558204, which are the same except for the strength of the constitutive lacI expression. We aim to specify the relation between the level of lacI production and the quality of IPTG induced expression. |
| [6] | BBa_K2558206 | CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI | This construct is the CRISPRi device, which is composed of dCas9, Ptac controlled gRNA targeting PHIF repressible promotor (BBa_K1725000) and constitutively (BBa_J23100, Anderson strong promotor) expressed lacI protein. We designed this device along with K2558207 and K2558208, which are the same except for the strength of the constitutive lacI expression. We aim to test the repression efficiency of our CRISPRi system with the help of PHIF repressible promotor. |
| [7] | BBa_K2558207 | CRISPRi device with Ptac expressed gRNA and Anderson medium (J23110)- expressed lacI | This construct is the CRISPRi device, which is composed of dCas9, Ptac controlled gRNA targeting PHIF repressible promotor (BBa_K1725000) and constitutively (BBa_J23110, Anderson medium promotor) expressed lacI protein. We designed this device along with K2558206 and K2558208, which are the same except for the strength of the constitutive lacI expression. We aim to test the repression efficiency of our CRISPRi system with the help of PHIF repressible promotor. |
| [8] | BBa_K2558208 | CRISPRi device with Ptac expressed gRNA and Anderson weak (J23114)- expressed lacI | This construct is the CRISPRi device, which is composed of dCas9, Ptac controlled gRNA targeting PHIF repressible promotor (BBa_K1725000) and constitutively (BBa_J23114, Anderson weak promotor) expressed lacI protein. We designed this device along with K2558206 and K2558207, which are the same except for the strength of the constitutive lacI expression. We aim to test the repression efficiency of our CRISPRi system with the help of PHIF repressible promotor. |
| [9] | BBa_K2558211 | lux pR test device | This construct is composed of constantly expressed luxR, and lux pR (http://parts.igem.org/Part:BBa_R0062) controlled sfGFP as our reporter. We aim to use this construct to help us test lux pR system and AHL induction efficiency. We designed this device along with K2558212, which is the same except for the different properties of the lux pR and the lux pR-HS promotor. |
| [10] | BBa_K2558212 | lux pR-HS test device | This construct is composed of constantly expressed luxR, and lux pR-HS controlled sfGFP as our reporter. We aim to use this construct to help us test lux pR system and AHL induction efficiency. We designed this device along with K2558211, which is the same except for the different properties of the lux pR and the lux pR-HS promotor. |
| [11] | BBa_K2558214 | NEON lux positive feedback device with luxpR-HS promotor | This construct is the NEON positive feedback device, containing constantly expressed luxR, lux pR-HS (K2258001) controlled luxI, and lux pR-HS (K2258001) controlled GFP. Without AHL stimulation, the lux pR-HS promotor is repressed by the dCas9-gRNA expressed by Safety Catch system. Therefore, the luxI and GFP cannot be expressed. Upon the stimulation of AHL, the CRISPRi system in Safety Catch is inhibited, thus the lux pR-HS promotor is exposed, and then luxR-AHL complex can now bind to and activate lux pR-HS promotor. Therefore, luxI and GFP can now be expressed. Besides, this system will keep producing more luxI-AHL, which will form a positive feedback and increase the expression of GFP. |
| [12] | BBa_K2558215 | NEON CRISPRi Safety Catch device with lux pR promotor | This construct is the “Safety Catch” test system, which is composed of lux pR controlled lacI, constantly expressed dCas9 and IPTG inducible gRNA that specifically binds to lux pR promoter on "Neon" system. Without AHL stimulation, gRNA is produced at a basal level and inhibit luxI and GFP (promoter: lux pR) transcription. Upon the addition of AHL, lacI on Safety Catch is transcribed (here we need to mention that even though the lacI on the Safety Catch is also controlled by lux pR, the design of gRNA allows us to make it unaffected by CRISPRi, thus it can still be activated by the luxR-AHL complex.) and prevents gRNA synthesis, thus relieving inhibition on lux positive feedback system on Neon. We designed this device along with K2558216, which is the same except for the strength of expression and some other parameters. |
| [13] | BBa_K2558216 | NEON CRISPRi Safety Catch device with lux pR-HS promotor | This construct is the “Safety Catch” test system , which is composed of lux pR induced lacI, constantly expressed dCas9 and IPTG inducible gRNA that specifically binds to lux pR promotor on "Neon" system. Without AHL stimulation, gRNA is produced at a basal level and inhibit luxI and GFP (promotor: luxpR-Hypersensitive, or lux pR-HS) transcription. Upon the addition of AHL, lacI on Safety Catch is transcribed (here we need to mention that even though the lacI on the Sately Catch is also controlled by lux pR, the design of gRNA allows us to make it unaffected by CRISPRi, thus it can still be activated by luxR-AHL complex.) and prevents gRNA synthesis, thus relieving inhibition on lux positive feedback system on Neon. We designed this device along with K2558215, which is the same except for the strength of expression and some other parameters. |
[1] Gilbert LA et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61.
[2] iGEM parts: lacI-regulated, lambda pL hybrid promotor (http://parts.igem.org/Part:BBa_R0011).
[3] H A de Boer et al. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan; 80(1): 21–25.
[4] H A de Boer et al. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan; 80(1): 21–25.
[5] H A de Boer et al. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan; 80(1): 21–25.
[6] Gilbert LA et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61. & iGEM parts: PHIF repressible promotor (http://parts.igem.org/Part:BBa_K1725000)
[7] Gilbert LA et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61. & iGEM parts: PHIF repressible promotor (http://parts.igem.org/Part:BBa_K1725000)
[8] Gilbert LA et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61.& iGEM parts: PHIF repressible promotor (http://parts.igem.org/Part:BBa_K1725000)
[9] Koch B et al. The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiology. 2005 Nov;151(Pt 11):3589-602.
[10] Koch B et al. The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiology. 2005 Nov;151(Pt 11):3589-602.
[11] Koch B et al. The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiology. 2005 Nov;151(Pt 11):3589-602. Gilbert LA et al. & Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61 & Daniel R et al. Synthetic analog computation in living cells. Nature. 2013 May 30;497(7451):619-23.
[12] Koch B et al. The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiology. 2005 Nov;151(Pt 11):3589-602. Gilbert LA et al. & Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61.
[13] Koch B et al. The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiology. 2005 Nov;151(Pt 11):3589-602. Gilbert LA et al. & Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61.