Degradation of Cellulose and Hemicellulose
The plant cell wall is composed of polysaccharides; cellulose, hemicellulose, and pectin (in order of abundance) [17] and lignin.
The most famous and abundant polysaccharide is cellulose or β-1,4-glucan, in both primary and secondary cell walls. The cellulose content in cell walls vary but can be up to 45% in particular primary cell walls[17]. It is a linear polymer made up β-1,4-linked D-glucose residues, existing in four polymorphic crystalline forms and is closely linked to xylan (a hemicellulose). In the cell wall, monomers are ordered to become fibres to give rigidity to the cell wall. Additionally, there are two types of cellulose; the native type and regenerative type[18]. To breakdown cellulose through Trichoderma and Aspergillus species, three main classes of enzymes are involved: endoglucanases (EG), cellobiohydrolases (CBH) and β-glucosidases (β-GD). EG hydrolyses cellulose to glucooligosaccharides followed by CBH degrading crystalline cellulose to produce cellobiose. Finally, β-glucosidase degrade the oligosaccharides to glucose[19]. However, as a results of different enzyme studies, exoglucanases have been seen to release glucose from cellulose and glucooligosaccharides however, it is not a clear distinction from the role of cellobiohydrolases[20]. Lastly, expression of cellulolytic genes in Aspergilli is observed in the presence of various monomeric and polymeric carbon sources.
Hemicelluloses are heterogeneous polysaccharides. They are used as a flexible cell wall support for plants and are able to bond to cellulose microfibrils through hydrogen bonding[13, 21]. However, the composition of this polysaccharide is different between plants and between species. There are many types of hemicelluloses but predominant types are xylan (in cereals) and xyloglucan (in onions). Xylan is a polymer consisting of a β-1,4-linked D-xylose backbone and a side group. In wheat straw, the side group to the xylose backbone are D-glucopyranosyluronic acid at position 2 and L-arabinofuranosyl and D-xylopyranosyl groups linked at position 3 on the backbone[22]. To degrade the xylan backbone, endoxylanses are used to cleave the backbone down to oligosaccharides and further degradation by β-xylosidases produces xylose [20]. For the degradation of xyloglucan, EGs and β-GD are used.
Finally, pectins are a group of heteropolysaccharies. The backbone of pectin is compost of α-1,4-linked D-galacturonic acid residues[20].
A non-polysaccharide component of the cell walls is lignin. In trees, lignin’s role in the cell wall is to support xylem cell and is covalently link to hemicellulose [23, 24]. However, in terms of advanced biofuel production, one of the key issues with the breakdown of wheat straw is lignin as it cannot be hydrolysed and monolignals are not involved to bioethanol production. Consequently, pre-treatment is a crucial step to allow access to cellulose and hemi-cellulose[25] which can be thermal, chemical or fungal. A chemical pre-treatment is to degrade lignin in wheat straw, is using a mixture of acetic acid–nitric acid or using white rot fungi [26, 27]. An aim for future biofuels is to reduce the amount of pre-treatments required since stronger pre-treatments reduce the availability of polysaccharides.
cbhB and cbhA
Cellobiohydrolase (CBH) A and B are involved in the breakdown of β-1,4-glucan down to glucose, particularly the breakdown of the crystalline cellulose. These two enzymes are encoded by the genes cbhA and cbhB respectively and belong to the fungal family CBH 7. The modular structure of cbhB contains a cellulose binding domain or carbohydrate binding module (CBM) domain which is linked to a catalytic domain by a Pro/Ser/Thr-rich linker peptide but cbhA has just the catalytic domain[28]. The modular structure of CbhB can be seen across a number of species and in T.reesei, over half of the protein secreted is cellobiohydrolase I (CBHI) which has the exact same structure is seen cbhB[11]. It must be noted that the lack of a CBD in cbhA only affects its cellulase activities with insoluble cellulose but not with other soluble substrates[29].
The regulation of many cellulases is at the transcriptional level. Transcriptional repression of these genes can be seen in T.reesei in the presence of glucose by CreA whereas transcriptional activation is induced by XInR[30]. Furthermore, the expression of cbhA and cbhB are activated by XInR, xylanolytic transcriptional activator in the presence of D-xylose.[28]
Carbohydrate-Binding Module (CBM) Domain
The CBM domain catalyses the inefficient attack on glycosidic bonds of polysaccharides by GHs[31]. Glycosidic bonds do not always fit in the active site of GHs therefore to overcome this, many GHS use catalytic and non-catalytic CBMs to promote their association with their substrate.
CBMs are mainly involved in to the hydrolysis of plant structure polysaccharides such as cellulose and hemicellulose. They also contain protein domains within a carbohydrate-active enzyme which is separate from a catalytic domain with carbohydrate binding activity[32]. A.niger has a subtype of CBM called the starch binding domain (SBD) which is found at the many amylolytic enzymes.
The CBM domain was previously defined as cellulose binding domain (CBD) because initial studies on these domains found these modules bound to cellulose [33, 34]. However, it can now be seen that modules appear to be bound to other carbohydrates. CBDs, in particular in T.reesei, are essential in cellulases performing the beginning steps of cellulose degradation as most of the substrate is still insoluble. However, not all cellobiohydrolase contain a CBD, such as cbhA in A.niger [28, 35].
Expression of Xylanolytic Enzymes
Xylanolytic enzymes breakdown xylan into xylose and are produced on xylose (monosaccharide), xylan (polysaccharide) or substrates containing these sugars[20]. However, xylanolytic enzymes are not induced by other monomeric or polymeric substrates such as glucose and cellulose. Some are cellulases are induced by xylose suggesting the presence of xylose may activate transcription genes encoding cellulases[28]. There is a separate regulatory control of synthesis of cellulases and beta-xylanases. In certain species such as A.terreus, xylanolytic enzyme, β-xylanase was induced by cellobiose and cellulose (which are structurally related to xylobiose and xylan) as well as a heterodissachride of glucose and xylose[36] [37]. Studies of the genes encoding xylanolytic enzymes (from A.niger and A.tubingensis) have shown that these enzymes are expressed in the presence of D-xylose, xylobiose, or xylan by XInR (transcriptional activator) however, when xylose concentration is too high or glucose is present, thee genes are repressed by CreA(catabolite repressor protein)[38-40].
XInR
A.niger has a transcriptional activator which regulates the expression of xylanolytic and some cellulolytic enzyme. The xInR gene encodes an 875 amino acid polypeptide which contains a zinc binuclear cluster domain and belongs to the superfamily of GAL4 transcription factors[41]. The binding site for XInR is 5′-GGCTAAA-3′ (confirmed in vivo and in vitro) and is a consensus sequence found in various Aspergilli and Penicillium chrysogenum. In filamentous fungi, the induction of xylanolytic genes is through a cis-acting element. The transcription factor affects the gene expression of enzymes involved in the degradation of xylan, arabinan and cellulose, including genes encoding endoxylanases (xInB and xInC) and beta-xylosidases (xInD) [42]. It was also found to regulate the expression of two endoglucanases; eglA and elgB. In terms of cellulolytic degradation[42], XInR has been shown to regulated the expression of α- and β-galactosidase genes (aglB and lacA) as well as cbhA and cbhB, two cellobiohydrolase genes[28, 43]. However, genes containing the XInR binding site within its promoter sequence are not automatically regulated by XInR, an example being endo-beta-1,4-glucanase A gene in A.nidulans, where gene expression was not detected in the presence of xylose[44].
Carbon Catabolite Repression
Catabolite repression in Aspergillus is mediated by a repressor protein, CreA at the transcriptional and post-transcriptional level [45, 46]. The protein, CreA, has a zinc finger motif which allows binding to target gene promoter sites containing SYGGRG[47]. A similar carbon catabolite repressor protein is seen in T.reesei called CRE1 and is 46% similar to CreA in A. nidulans[48].
CreA repression occurs on glucose, xylose and other carbon sources. If A.niger is carbon-starved, the addition of glucose to media causes transcription of creA genes within minutes but if carbon sources are low, transcription levels of creA are down-regulated by itself[46]. At low concentration levels of xylose, XInR activates genes encoding CHs however if xylose concentration increases to above 1mM, CreA-mediated repression takes place[49]. Nevertheless, the effect of high xylose concentration is weaker than glucose in terms of catabolite repression[20]. CreA does not just affect the expression hemicellulolytic enzymes but also arabinose catabolic enzymes[50].
Aim
This study aims to determine the gene expression of An01g11670 (eglA), which contains a CBM domain and An03g06550 (glaA) when Aspergillus niger is grown wheat straw, model carbon source, through a carbon (straw) timeline and a no carbon timeline. By comparing the expression of these proteins with control proteins (cbhA, cbhB and yefC) through a process of RNA extraction, RT-PCR and gel electrophoresis, it will be possible to deduce the responses of these proteins under the specified conditions.
Materials and Method
Growth Conditions for Aspergillus niger
Potato dextrose agar (PDA) (Oxoid), premade in house, was heated until melted. 100ml of the liquid PDA poured into 8 labelled tubes with the date and allowed to cool in an airing cupboard.
The A.niger strains used were N402 (ref) and maintained on potato dextrose agar (Oxoid). Slopes were incubated at 28oC until the cultures had conidiated and there was a thin layer of spores across the slope. Spores were resuspended in 0.1% (v/v) Tween 20 (Sigma). The cultures were inoculated with 106 spores/ml and grown in 100ml of minimal medial (see below for recipe). The appropriate carbon source was added to a final concentration of 1 % (w/v) in 250 ml conical flasks at 28 °C and shaken at 150 r.p.m.
For Carbon Timeline
The time course for the A.niger spores consisted of 48 hours of growth in 1 % (w/v) glucose media (as a carbon source) which allowed a mycelial mass to be formed. Mycelia were removed by filtration (Nalgene and MERCK), washed with water and transferred to straw media which contained 1 % (w/v) ground wheat straw (as a carbon source) and no glucose. 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. For the carbon timeline, Glucose was then added exogenously to a final concentration of 1 % (w/v) (S+G) and incubation continued for 6 hours. This was also extracted for RNA extraction.
For No Carbon Timeline
The time course consisted of growth for 48 h in 1 % (w/v) 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.
cDNA synthesis
For the cDNA synthesis mixture: 500ng of extracted RNA (Nanodrop Spectrometer, SLS) from each time point was added with 1µl of 50µM oligo (dt)20 (Invitrogen), 1µl of 10mM dNTP mix (10mM each of dATP, dGTP, dCTP, dTTP at neutral pH, in house) and water was added to make up to a final volume of 15µl. The tube (Invitrogen) was heated on heat block (Grant) for 5 minutes at 65oC, placed on ice for 5 minutes then spun down (Spingene, SLS). Finally, 4µl of 5x First-strand buffer, 1µl of 0.1M DTT and 1µl of Superscript III RT (200 units/µl) was added to the tube. Using the PCR machine (Techne TC-512), the contents of the tube was heated at 50oC for 60 minutes then 70oC for 15 minutes to deactivate the enzyme.
PCR
For 19µl master mix: 0.5µl of dNTP mix (10mM each of dATP, dGTP, dCTP, dTTP at neutral pH, in house), 4µl of 5X Phusion HF Buffer (NEB), 0.2µl of Phusion® High-Fidelity DNA Polymerase (NEB), 0.5µl of 500nM of the appropriate forward primers (Sigma), 0.5µl of 500nM of appropriate reverse primer (Sigma) and finally 1µl of the appropriate cDNA [Glucose/Initial, 0.5, 1, 2, 3, 6, 9, 12, 24, S+G, gDNA] was added. The tube then went in to the thermocyclers (Techne TC-512) and underwent the cycle shown below:
98oC - 2 minutes for denaturation
98oC – 20 seconds for initial denaturation
30 cycles
60oC - 20 seconds for DNA separation 72oC - 20 seconds for primer binding
72oC – 5 minutes for extension
10oC – for cooling
Forward and Reverse primers required: An07g02650 (yefC), An01g11670 (eglA), An03g06550 (glaA), cbhB, cbhA (all from Sigma).
To prepare the time point samples for loading into the wells, 5µl of 5x loading dye (in house) was added. After mixing, 5µl of each sample was taken out to be placed in the wells.
Gel Electrophoresis
Gels were prepared with the agarose (Lonza, Seakem®) content of 1.5% and mixed in solution with TAE buffer (in house). The standard gel used was 100ml of TAE buffer with 1.5g of agarose which was heated till a clear solution was seen. Once the mixture was cool to touch (5-10 minutes), 10µl of 10,000 x at 1mg/ml ethidium bromide (in house) was added before the mixture was poured into the insert with a 16 toothed comb. The gel was then left to set until a cloudy colour was seen. Gel and insert were placed into the tank (Bio Rad) with the comb removed and 600ml of TAE buffer was added. Finally, after the 5µl samples had been added to each well and 5µl of 100bp ladder (NEB) was added on the first well, the gel was run for 20 minutes at 100V using a power pack (Bio Rad).
The gels were imaged using Bio Rad Gel Cloc machine.
Restriction Enzymes (RE) Test
The check the PCR product size was correct; each gene was cut once with an applicable restriction enzyme. Once the genes had been amplified through standard PCR, 10µl of amplified cDNA was added to the RE mixture. For a final volume of 50µl RE mixture: 10µl of PCR product, 1µl of restriction enzyme (NEB); 0.5µl of BSA, if applicable or water (NEB); 5µl of NEB Buffer (NEB), 33.5µl of distilled water. The specific enzymes and buffer used can be seen in table 1. The RE mixture then went on a heat block at 37OC for one hour. This was followed by standard gel electrophoresis.
Table 1. The specific requirements for each gene for the Restriction Enzyme Test.
*All restriction enzymes, buffers and BSA are from NEB.
Primer PCR Test
To ensure the primers being used were binding to and amplifying the correct sequence during the PCR, glaA underwent standard PCR but the composition of PCR tubes are seen in table 2. The template used was standard genomic DNA (in house) and not cDNA produced before.
Table 2. Composition of the PCR tubes for the Primer PCR Test to ensure correct the correct PCR product size was produced.
This entire method section was based off the work of Stéphane Delmas and Steven T. Pullan[2]
Results
Carbon/Straw Timeline
The gene size for the cbhA (without introns) is 475bp and the bands present are between the 400bp and 500/575bp marks on the ladder. The expression of cbhA is induced at 9 hours. Between 9 and 24 hours, cbhA expression is at a consistent level but after glucose is added to the media, the level of expression has decreased (figure 1A).
The gene size for the cbhB (without introns) is 208bp and the bands present are close to the 200bp mark on the ladder. The expression of cbhB is weakly induced at 6 hours. From 9 hours until the end of the time course, cbhB expression is maintained at a constant level, even after glucose is added back into the solution (Figure 1B).
In figure 2, the expression of eglA is induced at 9 hours and maintained up to 24 hours. After glucose has been added, the expression of eglA appears to have decreased. eglA gene expression seems to correlate with the expression of cbhA. The gene size for the EGLA is 201bp and the bands present are close to the 200bp mark on the ladder (Figure 1A).
Expression of yefC (figure 1C) and glaA (figure 3) are mostly the same. Expression of yefC is not affected if A.niger is undergoing starvation and has a carbon source available for breakdown as it is a known house-keeping gene so its expression will be maintained regardless, of the nutritional status of the fungus. The gene size for the yefC is 717bp and the bands present are close to the 700bp mark (figure 1C). glaA’s gene size is 241bp (without introns) and all bands are between the 200bp and 300bp (figure 3).
No Carbon Timeline
When A.niger has wheat straw as carbon source, it is able to hydrolyse the polysaccharides in the plant cell walls to produced glucose as a food source. However, if there is no carbon source, the induction of may be genes may to be different.
When comparing figures 1A and 4A, the expression of cbhA is significantly lower when A.niger does not have a carbon source. A basal level of expression of cbhA is seen throughout the time-course as cbhA expression is not induced through starvation and is under the control of XInR.
In figure 4B, partial induction of cbhB is seen at 3 hours and the gene is fully induced at 6 hours. From 6 hours till the end of the time-course, its expression level does not change until 24 hours where expression has decreased.
Figure 4C shows that expression of yefC is at a consistent level throughout the time-course. This is the same for glaA (figure 6).
In figure 5, induction of eglA is seen at 6 hours and is maintained until the 12 hour time point but, expression levels at 24 hours have dropped.
All the band sizes in all figures correspond to the correct size of each gene.
PCR Restriction Digest Gels
The restriction digest of eglA and glaA were unsuccessful. In figure 10, there is one band present at around 250bp which close normal gene size of eglA (299bp) with introns. In figure 11, there is only one band present just above the 200bp marker which represents the gene size of glaA (201bp).
Figure 7 shows that cbhA was successfully cut by MfeI into its two fragments as two bands are present representing the 145bp and 330 fragment. In figure 8, two light bands representing the 50bp and 158 fragments from the MfeI digestions of the cbhB. Partial digestion of yefC is seen in figure 9. There are three bands present representing the uncut yefC at 717bp and the two cut fragments of yefC, 565bp and 152bp.
PCR Primer Test
Figure 12 shows the positive control, which contains both primers and template, was successful as a band size of 299bp is present. The negative control (No gDNA) shows that the PCR was not contaminated by other DNA or RNA. The lanes with just one type of primer show that PCR product size of just the forward primer above 400bp and of the reverse primer is just under 400bp. Therefore, if in the other figures, the PCR product size of glaA is around the 400bp mark, one of the primers may not have been correctly placed into the PCR tubes.
Discussion
By measuring the levels of RNA in A.niger, changes in gene expression at the transcriptional level can be seen. From the results, there is a clear induction of certain genes as a response from A.niger being starved. The presence of wheat straw is able to show if certain genes were under the control of XInR and if gene expression was mediated under CreA. It has been previously shown that CAzy gene transcription takes up 3% of the total mRNA after 48 hours of growth in 1% glucose[2]. CAzy genes refer to cbhA, cbhB, eglA and glaA.
Controls (yefC, cbhA, cbhB)
A.niger cbhA contains the XInR binding site within its promoter sequence and is therefore under complete control of the transcription factor, XInR [28]. In the presence of a carbon source (which contains hemicellulose) xylan degradation occurs so XInR induces cbhA transcription at 9 hours (figure 1A). Catabolite repression of glucose can be seen at the S+G time point as CreA represses the level of cbhA expression. When comparing the level of expression in between the two timelines, a basal level of expression of cbhA is seen without a carbon source suggesting that cbhA is not being transcribed (figure 1A and 4A). When there is no xylose or carbon source, XInR is not activated because there is no complex polysaccharide to degrade therefore, genes under the control of XInR are not induced (figure 4A). Figure 1A and 4A shows bands present at the 475bp. This shows that A.niger is able to degrade wheat straw if the solution it is contained in has no CreA stimulants such as glucose.
Figure 1B and 4B show that cbhB expression is only partially under the control of XInR. XInR induces hemicellulolytic genes in the presence of a carbon source hence the positive effect on the transcriptional level of cbhB seen in figure 1B at the 6 hour time point. However, if XInR was completely controlled cbhB, there would be no expression shown in figure 4B. This reaffirms previous studies showing that XInR only partially controls cbhB[2]. The results are not able to show this but cbhB is under the control of CreA[51].
yefC is a known housekeeping gene which encodes for a translational elongation factor 3 (An07g02650) [12, 52]. Housekeeping genes are involved in maintenance metabolism and are required to remain on, regardless of environmental conditions to A.niger[53]. Expression of yefC is not affected under stressful condition for A.niger as level of expression in both timelines remains constant (figure 1C and 4C).
eglA (An01g11670)
eglA encodes for endoglucanase A (EglA), a 440 amino acid polypeptide that is part of CBM Family 1 (from CAzy database) and GH Family 12. Endoglucanase are involved in the glucan degradation to glucose and EglA in particular, is highly specific to beta-glucan[54]. EglA has, as its family name suggests, a CBD mediated by three aromatic residues and therefore is involved in hydrolysing O-glycosyl compounds. CBM Family 1 modules are exclusive to fungi and have approximately 40 residues. In addition, EglA also has cellulase and cation binding activity. In A.niger, eglA is expressed when grown on sugarcane bagasse [55] as well as in response to wheat straw as shown in this study. XInR activates the expression of eglA and eglB in the presence of xylose, not sophorose [28, 42].
Both figures 2 and 5 show a band present 200bp representing the PCR product of eglA at 201bp; with introns, the gene size is 253bp. The transcription pattern of both eglA and cbhA are very similar; induction at nine hours and repression after glucose is added into the media ( figure 1A and 2) [28]. However, without wheat straw, expression of eglA is turned on earlier at 6 hours and stayed highly induced till 24 hours (figure 5) suggesting that expression of eglA must be induced by starvation. In figure 5, there is an anomaly in the band pattern because the band at the 24 hour time point should still show eglA being highly induced[2]. The cause of this anomaly is most likely due to not enough cDNA being synthesised.
glaA (An03g06550)
The glaA gene encodes for a 640 amino acid glucoamylase which has two alternate names; 1,4-alpha- D-glucan glucohydrolase and glucan 1,4-alpha-glucosidase and is responsible for 65% of CAzy gene transcription[2]. GlaA is involved in starch and sucrose metabolism to release glucose from the terminal ends of α-1,4-glucan subsequently glaA is expressed in high levels of starch[56, 57]. In terms of the CAzy database, glaA is placed in CBM20 and GH15 families. Specifically, glaA has a starch-binding domain unlike cbhB or eglA which have CBMs specific for cellulose[58]. Expression of glaA is under the control of the promoter, PglaA, the transcriptional factor AmyR and CreA[53, 59]. Furthermore, It has been shown that overexpression of glaA gene results in an increase in glaA mRNA and in extracellular production of GLA[60]. Finally, in the food industry, A.niger glucoamylase plays an essential role for the modification of starch[61].
Figure 3 and 6 both show that the level of glaA expression is not affected by the presence of wheat straw and is constantly on. As glaA expression is very similar to yefC (figure 1C and 4C), it would be easy to assume that glaA is also a housekeeping gene. However, glaA is not under the control of XInR but is AmyR-dependant which has been shown to be induced by D-maltose and D-glucose and repressed by xylose[56, 59]. 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 ΔxInR and ΔcreA 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 ΔxInR 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 ΔcreA 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.
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Appendix
RNA Test
This is the control test to ensure that was no DNA contamination and that there was RNA present.
by
teacher101 (student)
