Team:Tsinghua/Basic Part
Basic Parts
| Number | Name | Short Description | Long Description |
|---|---|---|---|
| [1] | BBa_K2558001 | lux pR-HS | This part is derived from lux pR. They are similar except for one point mutation in the luxR binding site (G19T) and another three in the -10 region of the promotor (TAG44CTT). The function remains the same, the mutant lux pR promotor is still inducible by LuxR/AHL complex. However, they do differ in gene expression strength, leakage and other parameters. |
| [2] | BBa_K2558003 | dCas9 | Cas9 is the main protein of the CRISPR/Cas system II of Streptococcus pyogenes. CRISPR systems protect bacteria and archaea from phages by recognizing and cleaving of invading phage DNA. In practice, a short gRNA is used as a guide and bind the Cas9 protein to its complementary DNA strand. Cas9 protein would cleave the DNA strand at the binding site. dCas9 is a mutant of Cas9 that lacks DNase activity. dCas9 preserves the ability to bind gRNA and then to gRNA’s complementary DNA strand, but does not cut DNA double strands. Therefore dCas9/gRNA complex is able to inhibit expression without damaging DNA. |
| [3] | BBa_K2558004 | Tac promotor (trp/lacUV5 hybrid) | Tac promotor (trp/lacUV5 hybrid) is a hybrid between Trp and lacUV5 promoters. It is a kind of strong E. coli promotor and is inducible with IPTG. |
| [4] | BBa_K2558005 | superfolder GFP | Superfolder GFP is a more robustly folded version of GFP, which is derived from GFP that often misfolds when expressed as fusions with other proteins. Superfolder GPF shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. |
| [5] | BBa_K2558006 | gRNA targeting PhIF promotor | gRNA targeting PhIF promotor is a sequence used in our CRISPRi device (K2558206, K2558207 and K2558208), whose function is to guide dCas9 to PhIF promoter and therefore inhibit the expression of downstream genes. |
| [6] | BBa_K2558007 | gRNA targeting luxpR and RiboJS | gRNA targeting lux pR and RiboJ is a sequence used in our NEON CRISPRi Safety Catch device (K2558214, K2558215), whose function is to guide dCas9 to the lux pR promoter and therefore inhibit the expression of downstream genes (luxI and GFP). Even though the lacI on the Safety Catch is also controlled by luxpR, this design of gRNA whose targeting sequence also includes part of the sequence of RiboJ allows us to make the lux pR in Safety Catch, which is not followed by RiboJ, unaffected by CRISPRi. Therefore lux pR on Safety Catch can still be activated by luxR-AHL complex. |
[1] 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.
[2] Gilbert LA et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell. 2014 Oct 23;159(3):647-61. & Qi LS et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28; 152(5): 1173–1183.
[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] Pédelacq JD et al. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006 Jan;24(1):79-88.
[5] iGEM parts: PHIF repressible promotor (http://parts.igem.org/Part:BBa_K1725000) & Qi LS et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28; 152(5): 1173–1183.
[6] Qi LS et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28; 152(5): 1173–1183.