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Gene expression in Aspergillus niger exposed to the lignocellulosic substrate, wheat straw

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Introduction

´╗┐Biochemistry Research Project (2012) Gene expression in Aspergillus niger exposed to the lignocellulosic substrate, wheat straw Shih-Han Chang (4102910) Biochemistry and Genetics BSc Supervisor: Professor David Archer Word Count: 6,959 ________________ Contents 1. Final Year Project Dissertation Declaration p4 2. Abstract p5 3. Introduction 1. Second Generation Biofuels p6 1. Bioethanol Production p7 1. Producing bioethanol from lignocellulosic biomass??????p7 1. Saccharification of lignocelluloses p8 2. Aspergillus niger p8 1. A.niger in response to starvation p9 1. Degradation of Cellulose and Hemicellulose p9 1. cbhB and cbhA p11 2. Carbohydrate-Binding Module (CBM) Domain p11 3. Expression of Xylanolytic Enzymes p12 4. XInR p12 5. Carbon Catabolite Repression p13 1. Aim p14 1. Materials and Method 1. Growth Conditions for Aspergillus niger p15 1. For Carbon Timeline p15 2. For No Carbon Timeline p15 3. Making Aspergillus Minimal Media solution p16 4. Making Straw Media for Carbon Timeline p16 1. Reverse Transcription Polymerase Chain Reaction p16 1. RNA extraction p16 2. Finding the Gene sequence CAZy p16 3. cDNA synthesis p17 4. PCR p17 5. Gel Electrophoresis p17 1. Restriction Enzymes (RE) Test p18 2. Primer PCR Test p19 1. Results 1. Carbon/Straw Timeline p20 2. No Carbon Timeline p22 3. PCR Restriction Digest Test p24 4. PCR Primer Test p26 1. Discussion 1. Controls (yefC, cbhA, cbhB) p27 2. eglA (An01g11670) p28 3. glaA (An03g06550) p29 1. Conclusion p30 2. Acknowledgements p31 3. References p31 4. Appendix p34 ________________ School of Biomedical Sciences Final Year Project Dissertation DECLARATION The work presented in this dissertation is my own work except where stated in the text. Technical assistance, where relevant, has been acknowledged. I understand the nature of plagiarism and that it is a serious academic offence. I confirm that no material in this project has been plagiarised. Name of Student ??????????????????????????.. (Please Print) Student Signature ??????????????????????????.. Name of Project Supervisor ?????????????????????. ________________ Abstract Over the past decade, the research on biofuel production has dramatically increased due to the increases in oil prices and carbon dioxide levels[1]. ...read more.

Middle

glucose media which allowed the A.niger to grow to its mycelia stage. Mycelia were harvest by filtration (Nalgene and MERCK), briefly washed with water and transferred to fresh AMM solution. Incubation was continued for another 24 hours. At selected hour intervals [Glucose/Initial, 0.5, 1, 2, 3, 6, 9, 12, 24] A.niger was extracted for RNA extraction. Making Aspergillus Minimal Media solution This solution does not contain carbon source (wheat straw). For a litre solution: NaNO3 (Sigma), 6 g; KCl, 0.52 g; MgSO4. 7H2O, 0.52 g; KH2PO4, 1.52 g; Na2B4O7.10H2O, 0.08 mg; CuSO4. 5H2O, 1.6 mg; FePO4.H2O, 1.6 mg; MnSO4.4H2O, 1.6 mg; NaMoO4.2H2O, 1.6 mg. The pH of the solution was maintained at pH 6.5 (Hanna pH meter instruments). If a carbon source was added, the solution was autoclaved at 1770C for 30 minute and if glucose was added, the solution was filter sterilised using Making Straw Media for Carbon Timeline Wheat straw (Sutton Bonnington) was added to the AMM solution to a final concentration of 1% (w/v) which was then autoclaved at 1770C for 30 minutes. The wheat straw was of the cordiale variety. It was composed of 37% cellulose and 32% hemicellulose and was ball milled using a Laboratory bill. Reverse Transcription Polymerase Chain Reaction RNA extraction A.niger spores were allowed to grow until the mycelia stage. At each time period, mycelia was extracted, frozen and ground under liquid nitrogen using a pestle and mortar. The RNA material extracted using the TRIzol reagent protocol (Invitrogen). An additional clean-up was done using the RNEasy Mini Kit (Qiagen), following the manufacturer's RNA Clean-up protocol, including the additional on-column DNAse digest. Finding the Gene sequence CAZy The sequence of each gene was found through Cadre-Genome website. The cDNA (without intron) sequence was exported from the website and using the primer sequence (forward and reverse), the replicated product produced (in PCR) was found. To the gene size, Primer Blast was used. ...read more.

Conclusion

Induction of glaA expression by glucose is stronger than that of xylose therefore, despite the wheat straw degradation seen at the carbon timeline, it must be noted that glucose is still a component of wheat straw and the xylose concentration was very low, consequently, glaA transcription remains constantly on (figure 3)[62]. ________________ Conclusion This study has shown that expression of CAzy genes can be affected by wheat straw at a transcriptional level. A.niger can be used as a model organism for the degradation of lignocelluloses as it is able to release specific plant cell-wall degrading enzymes. It is still unclear the precise mechanism by XInR and CreA modulate their expression therefore, experimentation in mutated strains of ?xInR and ?creA in 1% wheat straw timeline would show how they affect CAzy gene expression. Most of the CAzy genes (cbhA, cbhB and eglA) are XInR-dependant but glaA is under the control AmyR. In the ?xInR mutants, expression levels of glaA would still be the same as in wild type, cbhA expression will not be seen and cbhB may be partially seen. In ?creA mutants, expression of all the CAzy genes will be induced and not turned off. An additional experiment would be to do a low xylose (0.01%) timeline. This would provide further evidence of xylose induction of CAzy genes. Xylose induces of cbhA, cbhB and eglA expression but represses glaA. Both cbhB and glaA are induced by starvation therefore, induction should be seen at 6 and 9 hours respectively. With regards to glaA, there is contradicting research of xylose repression of glaA. An experiment with increasing xylose concentration would confirm if xylose has a repressive effect via CreA on the gene as well as confirm the precise concentration at which xylose repression is in effect. This experiment can also be applied to the other CAzy genes to further confirm the precise concentration for when CreA is active. ________________ Acknowledgements I would like to thank Matthew Kokolski, for his much appreciated assistance and contribution towards the study and Dr David Archer for his guidance. ...read more.

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