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Optimization of nucleic acid probes and transcriptional proteins for targeted data retrieval from DNA based data storage systems
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
2024 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Optimering av nukleinsyrasonder och transkriptionsproteiner för inriktad datahämtning från DNA-baserade datalagringssystem (Swedish)
Abstract [sv]

På grund av den snabba teknologiska utvecklingen så uppstår ett behov för alternativa metoder för storskalig informationslagring. DNA-baserade lagringssystem är ett lovande koncept med potential för långvarig lagring. Detta projektet ämnar att utforska lösningar som utnyttjar villkorlig transkription med hjälp av ett guide-protein och transkriptionsaktiverande molekyler; även användningen av T7 RNA polymeras och E.coli RNA polymeras promotorer kommer utforskas. De villkorliga proteinerna klonades med hjälp av E.coli DH5α celler, and uttrycktess i E.coli BL21-DE3 och E.coli T7 Express LysY/Iq celler. De kinetiska parametrarna mättes med In Vitro transkription (IVT). Efter transformation in i BL21-DE3 and T7 Express LysY/Iq celler så verkar de villkorliga proteinerna agera toxiskt  effekt och inhibera prolifirering. I allmänhet så verkar mutationerna på T7 promotorn förbättra bindningen men minska enzymets maxhastighet. För förbättrade och mer tillförlitliga resultat så bör natrium och monovalenta joner i allmänhet undvikas, och mer arbete bör investeras i att utvecklas en tillförlitlig matematisk model för att anpassa den generarade informationen. Gällande framtiden så verkar det dröja innan DNA-baserad datalagring kan konkurrera med dagens metoder. Däremot möjliggör det endast mer tid för att behandla de möjliga sociologiska och etiska dilemman som kan uppstå i konsekvens av utvecklingen av DNA-sekvensinriktade teknologier.

Abstract [en]

Because of rapid technological development, there is an emerging need for information storing technology with higher storing capacities. DNA-based storage systems is a promising system long-term storing potential. The proposed solution which will be explored in this project relies on conditional transcription through a guiding protein, and a transcription-protein-complex activating molecule; and the use of T7 RNA Polymerase and E.coli Holoenzyme promoters will also be explored. The conditional proteins were cloned using E.coli DH5α cells, and expressed in both E.coli BL21-DE3 and E.coli T7 Express LysY/Iq cells. The kinetic parameters were measured with In Vitro Transcription (IVT). The conditional proteins when transformed into the BL21-DE3 and T7 Express LysY/Iq cells seem to have a toxic effect and inhibit growth. In general, mutations on the T7 promoter seems to improve binding but reduce the maximum velocity of the enzyme. For improved and more reliable results, sodium and monovalent ions should be avoided, and more work should be invested in constructing a reliable computer model for fitting the generated data. With regards to the future, there seems to be long ways to go before DNA-based data storage is a viable contender to conventional methods. However, that only allocates more time for addressing the sociological and ethical issues that may emerge with the advancements of DNA-targeting technologies.

Place, publisher, year, edition, pages
2024.
Series
TRITA-CBH-GRU ; 2024:388
Keywords [en]
Information Storage, molecular biology, conditional transcription, protein kinetics, in vitro transcription, T7 RNA Polymerase, E.coli RNA Polymerase Holoenzyme, aptamer, promoter
Keywords [sv]
Informationslagring, molekylärbiologi, villkorlig transcription, proteinkinetik, in vitro transkription, T7 RNA Polymeras, E.coli RNA Polymeras Holoenzym, aptamer, promotor
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-356618OAI: oai:DiVA.org:kth-356618DiVA, id: diva2:1914676
External cooperation
Karolinska Institutet
Subject / course
Biotechnology
Educational program
Master of Science - Medical Biotechnology
Supervisors
Examiners
Available from: 2024-11-20 Created: 2024-11-20

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