Toward prototyping metabolic pathways in cyanobacteria using cell extracts

التفاصيل البيبلوغرافية
العنوان:	Toward prototyping metabolic pathways in cyanobacteria using cell extracts
بيانات النشر:	KTH, Proteinvetenskap 2022
تفاصيل مُضافة:	Bensabra, Amina
نوع الوثيقة:	Electronic Resource
مستخلص:	Cyanobakterier är intressanta mikroorganismer för produktion av biobränslen från solljus, vatten och atmosfärisk koldioxid och anses därför vara potentiella mikrobiella cellfabriker. Men på grund av långsam tillväxt och låg produktion är genteknologi processen intensiv och tidskrävande för cyanobakterier. En alternativ metod till prototypteknik för metabola vägar är att använda cellfri metabolisk teknik där cellysat av överuttryckta enzymer används. I detta projekt försökte vi utveckla en metod för cellfri metabolisk ingenjörsteknik för cyanobakterien Synechocystis PCC 6803 med hjälp av den övre mevalonatvägen som exempelreaktionsväg. Vi började med att utveckla tre fluorescensbaserade metoder för att detektera proteinöveruttryck med hjälp av de tre enzymerna från mevalonatreaktionsvägen. Dessa metoder använde fusering av YFP-proteinet till målproteinet, en delad GFP-reporterprotein eller translationskoppling. Ett av de överuttryckta enzymerna verkade vara giftigt för Synechocystis-celler så flera inducerbara promotorer användes för att försöka uttrycka enzymet. Den högst uttryckande konstruktionen för varje gen valdes ut och proteiner extraherades och blandades i en cellfri metabolisk ingenjörsreaktion. Även om inget mevalonat kunde detekteras med hjälp av gaskromatografi i detta projekt, berodde detta sannolikt på otillräckligt högt proteinöveruttryck av mevalonatgenerna. Cyanobacteria are desirable microorganisms for the production of biofuels from sunlight, water and atmospheric carbon dioxide, and are therefore considered potential microbial cell factories. But due to slow growth rate and low production rates, the engineering processes for bioproduction is labour intensive and time consuming. An alternative method to prototype metabolic pathway engineering is to use cell-free metabolic engineering, where cell lysates of enriched enzymes are used. In this project, we attempted to develop a method for cell-free metabolic engineering for the cyanobacterium Synechocystis PCC 6803 using the upper mevalonate pathway as an example pathway. We started by developing three fluorescence-based methods for detecting protein overexpression using the three enzymes from the mevalonate pathway. These methods used YFP fusion to target proteins, a split GFP reporter tag or translation coupling. One of the overexpressed enzymes appeared to be toxic to Synechocystis cells so several inducible promoters were used to try and express the enzyme. The highest expressing construct for each gene was selected and proteins were extracted and mixed in a cell free metabolic engineering reaction. Although no mevalonate could be detected using gas chromatography in this project, this was likely due to insufficiently high protein overexpression of the mevalonate pathway genes.
مصطلحات الفهرس:	cyanobacteria, metabolic engineering, cell-free metabolic engineering, enzymes, sustainability, cyanobakterier, metabolisk genteknik, syntetisk biologi, enzymer, hållbar bioproduktion, Biochemistry and Molecular Biology, Biokemi och molekylärbiologi, Student thesis, info:eu-repo/semantics/bachelorThesis, text
URL:	http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-315957 TRITA-CBH-GRU ; 2022:162
الإتاحة:	Open access content. Open access content info:eu-repo/semantics/openAccess
ملاحظة:	application/pdf English
أرقام أخرى:	UPE oai:DiVA.org:kth-315957 1348998686
المصدر المساهم:	UPPSALA UNIV LIBR From OAIster®, provided by the OCLC Cooperative.
رقم الأكسشن:	edsoai.on1348998686
قاعدة البيانات:	OAIster

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