Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Development of a shake flask method suitable for effective screening of Escherichia coli expression constructs
KTH, School of Biotechnology (BIO).
2011 (English)Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesisAlternative title
Utveckling av en skakkolvsmetod lämplig för screening av expressionskonstrukt i Escherichia coli (Swedish)
Abstract [en]

Screening of expression constructs suitable for protein pharmaceuticals is often done in batch cultivations. But the production of the recombinant protein is made during fed-batch cultivations. The two types of cultivations are different and therefore may good expression constructs that grow poorly in batch cultivations but good in fed-batch cultivations be rejected. Therefore would it be desirable to develop a fed-batch method that can be used in shake flasks. Biosilta has developed a method where starch is broken down into glucose by an enzyme creating fed-batch conditions. This method has been tried out and analyzed during this project. It is shown that the cells grown under these conditions can be glucose limited. However, at a later stage of the cultivation the cells produce a large amount of acetate and pH is not stable. The system builds on a booster tablet which content is unknown. If the booster is not added to the cultivations the cells stop growing, this indicates that there is some other limitation than just glucose. It is also seen that the amount of protein that is produced during this fed-batch mimic cultivation is much lower than that is produced during normal batch cultivations. I would therefore not recommend EnBase as a screening method.

Abstract [sv]

Screening av nya rekombinanta proteiner som ska användas till läkemedel sker oftast i batch-odling. Men själva odlingen av proteinläkemedlet sker sedan under fed-batch förhållanden. Dessa två typer av odling är olika och då cellerna växer olika kan detta leda till att fel, eller den inte mest lämpade kandidaten väljs. Därför vore det önskvärt att ta fram en fed-batch liknande metod i skakkolvar. Biosilta har tagit fram en metod där ett enzym bryter ned stärkelse till glukos som påminner om fed-batch. Denna metod har testats och undersökts i detta examensarbete. Det har visat sig att cellerna som växer under dessa förhållanden är begränsade på glukos men producerar stora mängder ättiksyra under den senare fasen av odlingen och att pH varierar mycket. Systemet bygger mycket på att en booster-tablett tillsätts, vad denna tablett innehåller är okänt. Men om tabletten inte tillsätts slutar cellerna att växa, detta tyder på att det finns någon mer begränsning än glukos. Det visade sig även att protein produktionen blev mycket lägre än vid odling i batch-fas. Det skulle av anledning av ovanstående inte vara bra att använda sig av EnBase som en screening metod.

Place, publisher, year, edition, pages
2011.
Keyword [en]
E. coli, expression, process development, screening, recombinant protein
National Category
Bioprocess Technology
Identifiers
URN: urn:nbn:se:kth:diva-49124OAI: oai:DiVA.org:kth-49124DiVA: diva2:459279
Educational program
Master of Science in Engineering - Biotechnology
Uppsok
Technology
Supervisors
Examiners
Available from: 2011-11-25 Created: 2011-11-25 Last updated: 2011-11-25Bibliographically approved

Open Access in DiVA

fulltext(1212 kB)663 downloads
File information
File name FULLTEXT01.pdfFile size 1212 kBChecksum SHA-512
14861df5a96424d44dcf913c6393ef1fe124316e8eb3183d23ba4232f110553a91bf639894fbba423a96f895c2b1b5b5451c33e827ae2ad0eeb72be560969ec3
Type fulltextMimetype application/pdf

By organisation
School of Biotechnology (BIO)
Bioprocess Technology

Search outside of DiVA

GoogleGoogle Scholar
Total: 663 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

urn-nbn

Altmetric score

urn-nbn
Total: 414 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf