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Team:OUC-China/Project Design
From 2014.igem.org
Transfection
Abstract
Why we use TAT
The TAT peptide (GRKKRRQRRRPQ) is derived from the transactivator of transcription (TAT) of human immunodeficiency virus and is a Cell-penetrating peptides. Cell-penetrating peptides (CPPs) have been used to overcome the lipophilic barrier of the cellular membranes and deliver large molecules and even small particles inside the cell for their biological actions. CPPs are being used to deliver inside cell a large variety of cargoes such as proteins, DNA, antibodies, contrast (imaging) agents, toxins, and nanoparticular drug carriers including liposomes.
Why we choose Histone H4
In previous studies, several groups found that histones can efficiently mediate gene transfer(histonefection). Histones Protein/peptide-mediated gene delivery doesn’t affect by serum.DNA delivery system can be inactivated by blood constituent,so the Efficiency of DNA Vector can’t be predictived,but Haberland verified the Histones Protein can successfully transfect into cell,even around the 100% serum. Histones Protein have strong NLS sigal. DNA plasmid and protein with NLS sequence, polypeptide or ipidosome can interact by electrostatic binding. Polypeptide contains NLS can covalently bond the DNA.H4 is strongest conservative,and has not found its subtypes yet. Choosing H4 protein as transfer gene carrier in targeted therapy has a relatively low immunogenicity, its security will be better.
Protection and transfection
We use NCBI to find the TAT sequence,we go through the paper and blast on NBCI,,finally confirm the Wheat Histone H4 DNA sequence,between the H4 and TAT we search the igem part registry ,because different types of linker have different function, so we finally choose the (Gly4Ser)3 Flexible Peptide Linker(BBa_K416001) .This is a 15 amino acid flexible peptide linker protein domain that is useful for creating functional fusion proteins. The linker is to be fused in frame in between two protein domains, separating the two domains so that they each retain original functions yet will be physically connected.The recombinant H4::TAT protein was expressed in E. coli .we design to test the fusion protein step by step,so we use the pET32a which can induce gene expression with IPTG and contains His-tagged ,so that Immobilized metal affinity chromatography(IMAC) was used to purify His-tagged fusion protein from supernatant protein by guanidine hydrochloride denaturation.The advantage of recombinant plasmid is condensing DNA and transfecting them into cultured cells efficiently. By changing the purpose of different genes can complete different targeted transport, this is a new type of transport carrier. Histones have high-density alkaline amino acid residues, When protein and plasmid are bonded to form stable compounds, According to the paper and the research,histones can pass through the plasma membrane of cells by passive diffusion.histones not only provide a new way to transport across the membrane,but also protect the plasmid from DNAase degradation.We design a plasmid with EGFP as reporter gene to verify wthether the recombinant plasmid complex could operate as we designed.
How to structure our lysis device?
To achieve the requirement that we need release the fusion protein and plasmid. This lysis device need to meet the following conditions: 1.The lysis device shouldn't do any harm to the TAT-H4 protein and plasmid. 2.The expression of leakage should be kept at a low level. If not, the bacteria will grow up slowly and the concentration of bacterium won't achieve our expectation. 3.The bacteria should lyse immediately and obviously after the induction. a holin protein causes "pores" in the inner membrane of E. coli, which allows lysozyme to access and break down the peptidoglycan in the periplasm, causing lysis. An antiholin molecule inhibits the activity of holin, and is used in the natural systems to control the timing of lysis. At present, we have structure two lysis devices, both of them are based on BBa_K112808（2008 Berkeley）.BBa_K112808 codes three kinds of protein from T4 phage lysis system. They are Holin, Lysozyme and Antiholin. The Holin protein causes "pores" in the inner membrane of E. coli, which allows Lysozyme to access and break down the peptidoglycan in the periplasm, causing lysis. An Antiholin molecule inhibits the activity of Holin, and is used in the natural systems to control the timing of lysis. To control the expression of lysis gene, we uses araBAD promoter (BBa_20600) and tet operon. The araBAD promoter will be induced by L-arabinose and tet operon will be induced by aTc. The circuit about tet operon and BBa_K112808 is as follow:
