Part:BBa_K4235010
mini-attTn7 segment: Tn7R+GmR circuit+Polyhedrin+PROS1+SV40+Tn7L
Introduction
Transposons (jumping genes) are DNA sequences that can change its location in a genome. These DNA sequences were first identified in the human genome by Barbara McClintock and transposons make up about 45-50 % of the human genome. Some common types of transposon elements are retrotransposons (Class 1 transposable elements), which use reverse transcription through an RNA intermediate to insert themselves back into a different location in the genome and DNA transposons (Class 2 transposable elements), which encode transposase, required for excision of the DNA sequence from one location and insertion into another location in the genome. Retrotransposons can be said to use a “copy and paste” mechanism for transposition, while DNA transposons use a “cut and paste” mechanism.
Roughly half of the human genome consisting of these transposable elements raises an important question, what roles do these DNA sequences play? Most of these transposable elements in the human genome are silenced, they do not actively move around in the genome and produce a phenotypic effect. This is either due to mutations in their sequences which hinder their ability to move around or they are rendered inactive by epigenetic mechanisms such as DNA methylation, modifying chromatin architecture and miRNA silencing. Many other reasons beyond epigenetics can regulate transposon activity in the human genome. However, in rare instances, transposons insertions into different locations in the genome can cause harmful mutations that may lead to disease development such as cancer. Transposons can also play a beneficial role in the evolution of genomes by shuffling of coding DNA sequences creating novel gene products, and also repairing double-stranded breaks.
Usage and Biology
Tn7 are terminal inverted transposon elements that flank both sides of the mini-attTn7 segment. These are used for site-specific transposition of heterologous gene circuits into attTn7 sites on a different chromosome and are more efficient in generating recombinant progeny than transposition through homologous recombination. Our project aimed at utilizing the mini-attTn7 segment for site specific transposition of our insert BBa_K4235000 from the pFastBac transfer vector onto the baculovirus genome (Bacmid), which is propagated in E. coli DH10Bac cells. Our mini-attTn7 circuit contains a Gentamicin Resistance gene BBa_K4235003, a multiple cloning site driven by a polyhedrin promoter BBa_K4235001, SV40 polyA signal and Tn7-L and Tn7-R ends for transposase activity. The Bac-to-Bac expression system exploits this mechanism of transposition to generate recombinant Bacmid particles in E coli DH10Bac, which can be isolated and used to infect insect cells for the production of recombinant proteins.
Bacmid DNA usually contains a kanamycin resistance gene and a lacZα coding DNA segment. The N-terminus of the lacZα gene contains a short segment (does not disrupt the reading frame of lacZα) , which serves as the attachment site for the bacterial transposon Tn7 or mini-attTn7 segment.
The initial step in generating recombinant bacmids is to clone the desired insert between the mini Tn7 elements of the mini-attTn7 segment in a pFastBac donor plasmid. Then the insert, flanked by mini Tn7 elements upstream and downstream can be transposed from the pFastbac donor plasmid onto the mini-attTn7 attachment site on the bacmid. The transposition function is provided by a helper plasmid already present in the E. coli DH10Bac cells, which expresses the enzyme transposase. Successful insertion into the bacmid will disrupt the expression of the lacZα coding segment. This allows for blue-white screening of successful recombinant bacmids using X-gal. Subsequent steps include isolating the recombinant bacmids, propagating, transfecting insect cells, protein expression and purification.
Mechanism of DNA transposition
DNA transposases are enzymes that recognize specific inverted terminal repeats (Tn7 elements) that flank both sides of the transposon sequence and facilitate the excision and movement of that DNA transposon mobile element. These mobile elements can be classified based on whether they encode the transposase enzyme or rely on transposase encoded by a different gene in the genome. Autonomous transposons themselves encode the transposase enzyme while non-autonomous transposons are recognized by transposases encoded at a different location.
There are a lot of biochemical mechanisms through which the enzyme transposase recognizes and binds to specific terminal repeats of the DNA transposon, introduce double-stranded breaks to free the remote mobile element and also identify the target DNA sequence, where the remote mobile element is meant to be inserted. Very detailed explanation of exact mechanisms of transposase activity can be found in Hickman, A. B., & Dyda, F. (2015).
References
- Hickman, A. B., & Dyda, F. (2015). Mechanisms of DNA Transposition. Microbiology spectrum, 3(2), MDNA3–2014. https://doi.org/10.1128/microbiolspec.MDNA3-0034-2014
- Pray, L. (2008) Transposons: The jumping genes. Nature Education 1(1):204
- Choi, KH., Gaynor, J., White, K. et al. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2, 443–448 (2005). https://doi.org/10.1038/nmeth765
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2914
Illegal XbaI site found at 2227 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2914
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2914
Illegal BglII site found at 37
Illegal BglII site found at 568 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2914
Illegal XbaI site found at 2227 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2914
Illegal XbaI site found at 2227 - 1000COMPATIBLE WITH RFC[1000]
//dna/transposon
//plasmid/chromosomalintegration
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