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Johnzon, Carl-Fredrik, 2011. Troubleshooting the GFP-tagging gene knockout (GGKO) method for the Leptosphaeria maculans effectors AvrLm6 and AvrLm4-7. First cycle, G2E. Uppsala: SLU, Dept. of Plant Biology and Forest Genetics (until 131231)

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Abstract

An attempt was made to GFP-tag the effector proteins of AvrLm6 and AvrLm4-7 using the GFP-tagging gene knockout method (GGKO) developed by Saitoh et al. (2008) in order to determine whether or not they are secreted. Successful pETHG-(target)KO vectors were not generated. The protocol was examined for potential errors. Fatal errors were pinpointed to the ligation reaction and the transformation required to generate and propagate the desired vector pETGH-(target)KO. The Downstreams Flanking Region inserts were evidently successfully ligated into the pETHG vector but for the Upstreams Flanking Region inserts the results were highly ambiguous. Due to a mistake, too high vector:insert ratios were used; altering them to the recommended 1:1 – 1:5 is hence expedient. It was reasoned that the bacteria possibly absorbed empty pETHG vectors instead of putative insert-carrying pETHG-(target)KO vectors during the transformation. The procedure could be improved by digesting pETHG twice prior to ligation as well by separating linearised and uncleaved pETHG by gel extraction. The latter could also be performed on the ligation product. Suggested general improvements include: Sequencing the PCR product and purifying it of potential restriction enzyme inhibitors, use the maximal incubation time for the restriction enzymes, expand the colony screening, increase the spectinomycin concentration and test different bacterial strains in the transformation.

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Ett försök gjordes att GFP-markera AvrLm6 och AvrLm4-7 proteinerna via ”GFP-tagging gene knockout” (GGKO) vektor systemet som utvecklats av Saitoh et al. (2008). Syftet var att utröna om genprodukterna från dessa gener återfinns inne i värdväxten under infektionens gång. Inga pETHG-(target)KO vektorer genererades under projektets gång. Via analys av resultat från rutinmässiga kontroller i experimentet samt tester av vissa steg begränsades de potentiella kloningsproblemen till transformeringsteget med viss tvekan med avseende på om uppströmsflankerande regionen ligerades till pETHG eller ej. På grund av ett misstag användes fel vektor:insert förhållande i ligeringsreaktionen. De rekommenderade 1:1 – 1:5 förhållandena bör således användas. För att förhindra den potentiella upptagningen av tomma pETHG under transformeringen, kan pETHG behandlas två gånger med restriktionsenzymen därtill kan linjära vektorer separeras från obehandlade vektorer via gel extraktion. Det senare kan även utföras på ligeringsprodukten. Allmänna förbättringar innefattar: Sekvensera PCR produkten samt avlägsna potentiella restriktionsenzym inhibitorer, använda den maximala inkubationstiden för restriktionsenzym, utöka koloni screeningen, höja spektinomycin koncentrationen samt testa olika bakteriestammar för transformeringen.

Main title:Troubleshooting the GFP-tagging gene knockout (GGKO) method for the Leptosphaeria maculans effectors AvrLm6 and AvrLm4-7
Authors:Johnzon, Carl-Fredrik
Supervisor:Peele, Hanneke
Examiner:Dixelius, Christina
Series:Independent project / Department of Plant Biology & Forest Genetics, SLU
Volume/Sequential designation:119
Year of Publication:2011
Level and depth descriptor:First cycle, G2E
Student's programme affiliation:NK002 Biotechnology - Bachelor's Programme 180 HEC
Department:(NL, NJ) > Dept. of Plant Biology and Forest Genetics (until 131231)
Keywords:Leptosphaeria maculans, AvrLm6, AvrLm4-7, GFP, GGKO
URN:NBN:urn:nbn:se:slu:epsilon-s-379
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-379
Subjects:Plant diseases
Research methods
Language:English
Deposited On:27 Jun 2011 13:12
Metadata Last Modified:20 Apr 2012 14:20

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