Team:OUC-China/Project Design

Transfection

Abstract

In order to complete the transfering of our plasmid into cells, we combined the TAT-H4 fusion protein with our target plasmid in the hope that the fusion protein will have the function of TAT-PTD and Histone H4. In this way, it can transfer our plasmid into the eukaryotic cell efficiently and completely. With the purpose of releasing TAT-H4 and plasmid outside the cells, we have constructed a device autolysis.

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 into the 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 isn’t affected by serum. DNA delivery system can be inactivated by blood constituent, so the Efficiency of DNA Vector can’t be predicted, but Haberland verified that the Histones Protein can successfully transfect into the cell, even in the 100% serum. Histones Protein have strong NLS signal. 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 strongly conservative, and has not found its subtypes have not been found yet. Choosing H4 protein as transfer gene carrier in targeted therapy has a relatively low immunogenicity, so its security will be better.

Protection and transfection

We used NCBI to find the TAT sequence and, we went through the papers and blast on NBCI. Finally we confirmed the Wheat Histone H4 DNA sequence, between the H4 and TAT. We searched the igem part registry, because different types of linker have different function, and we finally choose the (Gly4Ser) 3 Flexible Peptide Linker (BBa_K416001) because different types of linkers have different functions. 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 their original functions yet they will be physically connected. The recombinant H4::TAT protein was expressed in E. coli. We planned to test the fusion protein step by step, so we used the pET32a which can induce gene expression with IPTG and contains a 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, we can complete differently targeted transport, and 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 papers and the research, histones can pass through the plasma membrane of cells by passive diffusion. Histones not only provide a new method to transport across the membrane, but also protect the plasmid from DNAase degradation. We designed a plasmid with EGFP as reporter gene to verify wthether the recombinant plasmid complex could operate as we designed.

How to construct 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:

• Double Plamisd
  • Abstract
  • The Design Of Mini Plasmid
  • The Deactivation Of RP4 OriT Site
  • Test
• Transfection
  • Abstract
  • Why We Use TAT
  • Why We Choose Histone H4
  • Protection And Transfection
  • How to construct our lysis device