Banca de QUALIFICAÇÃO: Marco Antônio de Oliveira

Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.
STUDENT : Marco Antônio de Oliveira
DATE: 29/09/2023
TIME: 10:00
LOCAL: Plataforma virtual
TITLE:

"Assembly of serine-integrase controlled genetic circuits integrated into the minimized genome of the synthetic bacterium Mycoplasma mycoides JCVI-Syn3B and into the complex eukaryotic genome of and their applications in Synthetic Biology".

KEY WORDS:

Serine-integrase ; Genetic circuits; Genetic switch ; Minimal cell ; Mycoplasma mycoides JCVI-Syn3B ; Nicotiana benthamiana ; Synthetic biology

PAGES: 78
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

The ability to regulate gene expression in response to external cues is one of the central mechanisms of the differentiation and maintenance of life in nature, as well as one of the main goals of scientists in efforts to control and reprogram organisms. The availability of molecular tools that allow genetic manipulation is crucial for such advances in synthetic biology, especially when creating intricate genetic circuits and activation cascades to work as synthetic regulatory networks. Serine-integrases (Int) are strong candidates for these applications due to their properties, especially their orthogonality and ability to work without any additional cofactors. Moreover, depending on the placement of their attachment (att) sites, the recombination can result in different outcomes, including insertion, excision, or inversion of a target sequence. Taking advantage of their plasticity and robustness, here we propose the application of serine-integrases in the assembly of genetic circuits integrated into the genome of two models of opposing complexity: Mycoplasma mycoides JCVI-Syn3B, a synthetic bacterium hosting a minimized prokaryotic genome, and Nicotiana benthamiana, a model plant with its complex eukaryotic genome. In M. mycoides JCVI-Syn3B, Int9 or Int13 were first tested as effectors in genetic switches capable of inverting a reporter gene sequence, but only Int9 activation resulted in a reporter gene expression increase. Int9 was then selected as an activator for a switch controlling the expression of I-CeuI - an endonuclease used for digestion and removal of the cell’s genome for the production of Simple Cells (SimCells), a valuable platform for synthetic biology applications. Our Int9-based switch was able to tightly control I-CeuI expression. Finally, envisioning a kill-switch for selection of the SimCells generated, new inducible promoters were tested, with only the pA13/AraE+AraR promoter presenting a proper response so far, although yielding considerably low expression levels. As for N. benthamiana, a more complex switch was assembled. Named Int-Plex@ (Integrase-Plant Expression), this genetic switch system consists of a reporter mgfp gene sequence flanked by a combination of att sites of Ints Bxb1, PhiC31, Int 13, and Int9 and can be divided into two modules: the inversion module and the excision module. The inversion module is composed of Int9 and Int13 att sites and their recombination results in a 180-degree flip of the target DNA, leading to activation of mGFP expression. Concurrent or sequential activation of both Ints will bring the reporter gene to the initial silenced state. The excision module is activated by transient expression of Bxb1 or phiC31. In this case, the DNA sequence flanked by their attachment sites is irreversibly excised from the genome. Integrases were delivered via agroinfiltration, and all four Ints were able to edit N. benthamiana genome successfully. This memory switch system can be used in future genetic circuits to engineer and modulate plant metabolic pathways of economic and environmental importance, while also addressing transgene biocontainment issues given the possibility of cassette excision. Adding to the efforts for advances in synthetic biology, this work includes the implementation of protocols for the production and use of cell-free expression (TxTl) and encapsulation to produce synthetic systems. Int-Plex@ was shown to also work in TxTl reactions.

COMMITTEE MEMBERS:
Interna - 1208515 - ANDREA QUEIROZ MARANHAO
Presidente - ***.402.491-** - ELIBIO LEOPOLDO RECH FILHO - NOTTINGHAM
Externo à Instituição - JOÃO RICARDO MOREIRA DE ALMEIDA - EMBRAPA
Interno - 2122537 - MARCELO DE MACEDO BRIGIDO