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Team:OUC-China/Project Design
From 2014.igem.org
Double plasmids
Abstract
Our project aims at designing a plasmid, which can transfer among a wide range of bacteria species by conjugation. So that the engineered bacteria can transmit the functional plasmid into bacteria living inside animals natively if we inoculate our engineering bacteria into animals.
We use plasmid RP4 to create the condition for conjugation. Plasmid RP4 has the ability of transferring among most of gram positive bacteria and a portion of gram negative bacteria, such as Vibrio harveyi. But the plasmid carries many resistance genes. So in consideration of biosafety, we expect that the plasmids transferred into recipient bacteria no longer have the ability of transferring by conjugation. Therefore we designed a double-plasmid-system:
1. Plasmid RP4 creates the basic condition for conjugation, such as synthesis of DNA relaxase, ATP hydrolytic enzyme and other protein needed for conjugation.
2. Another plasmid carries functional genes, but only with the assistance of RP4 can it transfer by conjugation. We call it “mini plasmid”.
The design of mini plasmid
We get the oriT region of plasmid RP4 by means of PCR, and ligate it with the reporter gene BBa_J04450 coding, then a non-self-controllable conjugated part is constructed and we ligate the coding to pSB1C3. With the help of RP4 (with the oriT site deactivated), the recombinant plasmid could be a conjugative plasmid (the principle of conjugation has been mentioned in Background).
The deactivation of RP4 OriT site
We hope that the OriT site (Origin of transfer for the RP4-plasmid nic region) can be deactivated through homologous recombination, so that plasmid RP4 loses the ability of conjugation. However, RP4 still expresses the functional proteins that are needed in conjugation.
Test of conjugation
Our project regards the E.coli in zebrafish as conjugation mock objects. We isolated and sequenced the E. coli in zebrafish gut, and used it as recipient strain to do the conjugation experiment. Finally, we proved that the mini plasmid can enter into the E.coli successfully with the help of RP4. Because the related physiological and biochemical characteristics of the intestinal E. coli in zebrafish has not been studied well, we used type strains as experimental material, such as HB101 and Top10. (Please refer to protocol for experimental details)
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 TAT-H4 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 meet the demand of releasing the protein and plasmid, our self-lysis devices need to reach the following requirements:
1. The lysis won’t break down the TAT-H4 fusion protein or plasmid.:
2. The background expression should be kept at a low level. If not, the bacteria cann’t grow up to the expected concentration.
3. The engineered bacteria should lyse immediately and remarkably after induction.
We have constructed two self-lysis devices, both of which are based on BBa_K112808（2008 Berkeley）. BBa_K112808 codes three kinds of proteins of T4 phage lysis system, holin, Lysozyme and antiholin. 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. (2008 UC_Berkeley https://2008.igem.org/Team:UC_Berkeley/LysisDevice):
To control the expression of lysis genes, we used pBAD promote r (BBa_206000) and tetracycline operator. The pBAD promoter will be induced by L-arabinose and tetracycline operator will be induced by anhydrotetracycline (aTc). We combined the pBAD promoter or tetracycline operator with coding sequences of lysis proteins, so that we got two self-lysis devices, A and B as follows::
In consideration that antiholin molecules inhibit the activity of holin molecules, we take down the lysis coding DNA sequence without antiholin by PCR, hoping to realize more significant lysis. Combining it with the pBAD promoter or tetracycline operator, we constructed other two self-lysis devices, C and D as follows:
Reference
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