Бесплатный автореферат и диссертация по биологии на тему

Разработка и использование методов направленной модификации целевых генетических локусов хромосомы E. coli при конструировании штаммов-продуцентов аминокислот

ВАК РФ 03.00.03, Молекулярная биология

Заключение Диссертация по теме "Молекулярная биология", Каташкина, Жанна Иосифовна

выводы

1. На основе специализированного плазмидного вектора создана коллекция прокариотических регуляторных элементов (промоторов и двух р-независимых терминаторов транскрипции), узнаваемых РНК-полимеразой E.coli, и охарактеризована их относительная эффективность.

2. Проведено КОНСТруИрОВаНИе НОВЫХ Промоторов Р<ac-ideal И P(rc-ideal НЭ ОСНОВе Рtac и Рtrc., имеющих более высокий чем их предшественники уровень репрессии Lacl. Сконструирован новый "сильный" гибридный промотор Рь-гас> который репрессируется как Lacl, так и Я,С1.

3. Созданы и изучены свойства новых авторегулируемых генетических элементов на основе промоторно-операторных областей и генов репрессоров лактозного оперона E.coli и бактериофага X - РiacVy5-^lacI и Рi^clts857.

4. Разработан эффективный метод модификации бактериальной хромосомы с последующей эксцизией селективного маркера в результате использования Int/Xis- и Red-зависимых систем рекомбинации бактериофага X. Показано, что с помощью этого метода можно осуществлять как специфические делеции генов, так и более сложные реконструкции хромосомы, в частности, замену регуляторных элементов генов.

5. Разработана процедура оптимизации экспрессии генов, локализованных в хромосоме E.coli. При использовании гена lacZ в качестве репортера показана возможность значительного (не менее двух порядков величины) изменения уровня экспрессии гена путем замены нативной промоторной области на РНеподобные промоторы с вырожденной последовательностью нуклеотидов в области «-35». При этом получена коллекция фрагментов ДНК, содержащих селективный маркер - XattL-Cm^-XattR, соединеный с Р,^-подобными промоторами с известной относительной эффективностью инициации транскрипции.

Библиография Диссертация по биологии, кандидата биологических наук, Каташкина, Жанна Иосифовна, Москва

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