User:Radoslaw Kwasniak/Notebook/Transcription factor delivery through T3SS

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Synthetic biology is a field where engineering principles, such as parts abstraction and standardisation, interchangeability of components and relying on design are applied to biology. It aims to change biological experiments from empirical and focused on discovery, to outcome – oriented and to a greater extent predictable. Parts abstraction and standardisation is a process of identifying a useful function or molecule produced by an organism, obtaining the gene responsible for the feature or chemical, and modifying the DNA part to be compatible with other parts. The final DNA sequence is kept in a plasmid and is flanked by precise upstream and downstream sequences containing restriction sites for EcoRI, XbaI, SpeI and PstI endonucleases. This form of DNA part is called a BioBrick and it allows for interchangeability of the parts. Interchangeable parts are components that can be assembled into a genetic circuit (promoter, RBS, BioBrick, terminator) at random and be able to perform their function. This needs to be carried out within tolerances, as the function of one part is affected by the other parts. Different BioBricks work with varying efficiency depending on parts they are linked to. Parts standardisation, interchangeability and availability lead to accelerated progress in developing genetic constructs with increasing complexity, as the design of genetic circuit becomes the major part of the process, instead of the necessity to control reliability and correct function of the genes. With assured functioning of the parts, scientists can focus on higher horizons making synthetic biology highly applicable to solving world’s challenges. Type III secretion system (T3SS) evolved in bacteria as a way to introduce certain molecules into the host cells. Ubiquitous bacterium Pseudomonas aeruginosa uses this system to infect animal and plant host cells. One of the toxins the bacteria secrete through T3SS is exoS. The sequence of first 54 amino acids of the toxin was identified to be a signal for secretion through the T3SS needle. The functioning of this secretion signal was confirmed by Bichsel et al., (2011), who attached a nuclear protein to the signal and observed its effects on the DNA of the infected cells. In their publication the group suggested applicability of this approach in generating induced pluripotent stem cells (iPSCs). IPSCs are a type of stem cells generated by forced expression of certain genes in somatic cells. Combined action of Oct4, Sox2, Klf4 and c-Myc genes maintains the undifferentiated state and self – renewal of the cells. Currently the most common method of generating iPSCs involves using viruses as transgene vectors. In this method, introduced genes are incorporated at random in the DNA of the host cells. The drawbacks of this procedure are unpredictability and the risk of activation of oncogenes. The transcription factors, which are coded by those genes and are able to activate pluripotency genes on their own, can be delivered into the cells directly through bacterial type III secretion system thus providing a safer method for iPSCs generation. This project will explore this route by constructing a vector containing T3SS signal which will provide a means of delivery of a molecule of choice into host cells through bacterial T3SS. The vector will be constructed using BioBricks and will be made available to the Registry of Standard Biological Parts.


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