Level of CO2 that Affects the Microbial Activity, Diversity, and Numbers - Assignment Example

1. a. Describe what are the likely factors that may change during exposure of a system to elevated atmospheric CO2 which may affect soil microbial activity, diversity and numbers. Through some experiment it was noted that some factors affecting by the elevated level of CO2 that affects the microbial activity, diversity and numbers. Research results Microbial C:N ratios significantly decreased under elevated CO2 (– 15%, P = 0.01) and microbial N increased by + 18% (P = 0.04). Soil basal respiration was significantly increased on one out of 7 sampling dates (+ 14%, P = 0.03; December of the third year of treatment), whereas the metabolic quotient for CO2 (qCO2 = basal respiration/microbial C) did not exhibit any significant differences between CO2 treatments. Also no responses of microbial activity and biomass were found in a complementary greenhouse study where intact grassland turfs taken from the field site were factorially treated with elevated CO2 and phosphorus (P) fertilizer (1 g P m2 y1). Previously reported C balance calculations showed that in the ecosystem investigated growing season soil C inputs were strongly enhanced under elevated CO2. It is hypothesized that the absence of microbial responses to these enhanced soil C fluxes originated from mineral nutrient limitations of microbial processes. Laboratory incubations showed that short-term microbial growth (one week) was strongly limited by N availability, whereas P was not limiting in this soil. The absence of large effects of elevated CO2 on microbial activity or biomass in such nutrient-poor natural ecosystems is in marked contrast to previously published large and short-term microbial responses to CO2 enrichment which were found in fertilized or disturbed systems. It is speculated that the absence of such responses in undisturbed natural ecosystems in which mineral nutrient cycles have equilibrated over longer periods of time is caused by mineral nutrient limitations which are ineffective in disturbed or fertilized systems and that therefore microbial responses to elevated CO2 must be studied in natural, undisturbed systems. 2. a. What factors may influence the efficicency of DNA extraction from an environmental sample. The ratio of liquid volume to sediment volume influences lysis efficiency, the extract liquid volume used for all extractions was 900 μl. RNase will create problem Presence of humic acid in the DNA extracted fom the soil also a problem. b. What is the purpose of the GC clamp on one of the primers. (10 %) GC-clamp is a stretch of GC-rich sequences used in DGGE and TGGE. Also refers to a small number of G or C frequently used at the 3-end of a PCR primer such that the 3-end of a primer will form stable complex with the target DNA. So during the time of primer design GC content is very important c. What factors may affect the success of PCR of DNA extracted from soil. After DNA isolation from the soil sample it was always noted that the DNA solution contain humic acid also, it’s a type of contaminant. These compounds absorb at 230 nm whereas DNA absorbs at 260 nm and protein at 280 nm. To evaluate the purity of the extracted DNA, absorbance ratios at 260 nm/230 nm (DNA / humic acids) and 260 nm/280 nm (DNA / protein) were determined. So this impurity will create major problem during PCR reaction. Because PCR held only in the sterile condition so presence of impurity will lead the undesired fragment amplification or will not get good result. The bacterial fraction of soil was separated from the inorganic or humic layer by a differential centrifugation technique. Bacterial cells were lysed using lysozyme and the DNA purified using ammonium acetate precipitation and ethanol precipitation (14). DNA was re-suspended in TE. 3. a. What are the limitations of environmental fingerprinting technologies such as DGGE. What other techniques can be used. Denaturing Gradient Gel Electrophoresis A method for finding out single base changes in the genetic structuring of DNAs. Different types of DNA’s would proceed at varying speeds in a denaturing gradient gel for the primary reason that they have varying melting points and partially melted DNA move at lesser speed. In denaturing gradient gel, DNA is melted by large dozes of urea salt. DGGE gives “sequence dependent, size independent method”. To comprehend DGGE, there are two aspects to be considered. The first is how the composition of DNA varies with temperature and the second is how variances in composition of DNA affect the migration of DNA through gel. At room temperature, with the availability of, minimum, a mM of salt, the twin pieces are quite composed, and we can consider the molecule to be actually like two strings tightly bonded to one another. Thus, in effect, there are 2 ends. DNA is a negatively charged molecule (anion) and, on the face of an electric field, would proceed to the positive electrode. A gel is a molecular network with openings almost the same size as the diameter of the DNA string. In the presence of the electric field, the DNA would try to move through the mesh, and, based on a certain set of circumstances, the speed of its flight would roughly correspond to the length of the DNA molecule. This is the premise for size dependent, separation in standard electrophoresis. As the temperature rises, the two strings of the DNA would split, due to melting. At very high temperatures, it would split into two discrete parts. But at middle temperatures, it would be partially split and partially together. This is analogous to a string which could be split into a ‘Y’ formation by partial splitting or made into 4 ends, by splitting both ends partially. In DGGE, gradient of denaturing adding to the gel to denature the DNA. DGGE serves its purpose because the movement of the DNA molecule through the gel reduces significantly when the partially melted compositions are formed, and more importantly, the exact temperature at which this happens depends on the order. Thus, DGGE gives “sequence dependent, size independent method” for separating molecules. This is similar to a situation when a person moves in a crowded room, if he stretches out his hand, his movements would slow down significantly, even though the body mass has not altered. Denaturing gradient gel electrophoresis (DGGE ) works by applying small samples of DNA (RNA) to an electrophoresis gel that contains denaturing agent.Through research it has been proved that certain denaturing gels have the ability to make DNA to melt at different levels. As a consequence to this melting, the DNA moves through the gel, and can be examined for single components, even those as small as 200-700 Base Pairs. SHORTCOMINGS The method for obtaining finished gels that can be used for investigative research requires further procedures (such as amplification of the samples from the gel) Although the results obtained through DGGE are more accurate and genuine than previously tested methods, it suffers from certain innate defects. “Chemical gradients such as those used in DGGE are not as reproducible, are difficult to establish and often do not completely resolve hetero-duplexes (Westburg 2001). Due to the inherent defects of DGGE, researchers have now developed a more reliable and appropriate technique called TGGE, which is superior to DGGE. It follows the same principle as that of TGGE such as the use of melting properties of the molecules as the principle method for splitting the gel. However, the method differs in that white TGGE ‘provides a temperature gradient instead of a chemical gradient.” (Spanevello 1997) Read More