BIO 2290 Flashcards
What are biopharmaceutical applications of bacteria?
Bacteria are used to create proteins that are useful to us by transcribing and translating genes in plasmids.
How are bacteria used in transgenic organism creation?
Bacteria help in creating transgenic organisms by expressing foreign DNA.
What are cDNA libraries used for?
cDNA libraries are used for genotyping and phenotyping studies.
What is competent bacteria?
Bacteria that can take up exogenous DNA.
What features must an appropriate plasmid have?
It must be taken up and expressed in bacteria and allow for screening of bacteria with the plasmid and insert.
Why is the plasmid’s origin of replication (Ori) important?
It ensures multiple copies of the plasmid exist in each bacterial cell.
What is antibiotic resistance in plasmids used for?
It provides a selectable marker, ensuring only transformed bacteria survive on antibiotic plates.
Why identify restriction enzyme sites on a plasmid map?
To know where to insert DNA fragments.
What is the multiple cloning site (MCS)?
A region with a high density of restriction enzyme sites where DNA fragments can be ligated.
Where is the MCS located?
In the LacZ operon.
What does LacZ code for?
β-galactosidase, which creates a blue pigment when metabolizing its substrate.
How does the LacZ system indicate successful ligation?
White colonies indicate successful ligation, while blue colonies indicate no ligation.
What is the first step in plasmid insertion?
Digest both plasmid and DNA insert with the same enzyme.
How is recombinant plasmid introduced into bacteria?
By transforming competent E. coli and plating on antibiotic plates.
How are colonies prepared for plasmid isolation?
Pick colonies and incubate them in liquid LB overnight.
What technique is used to isolate plasmid DNA?
Mini prep
What is the role of restriction enzymes?
To cut DNA at specific sites.
What are sticky ends?
DNA ends cut in an offset manner, creating single-stranded overhangs.
What are blunt ends?
DNA ends cut straight across without overhangs.
Why are sticky ends more efficient for ligation?
They provide complementary base pair attraction.
What is the issue with single/blunt digests in ligation?
The insert can go in any orientation, which may disrupt proper expression.
How does a double digest address orientation?
It creates two different sticky ends, ensuring the insert can only go in one orientation.
Why is the insert-to-vector ratio important?
It affects ligation efficiency and must be optimized (e.g., starting at 3:1).
What is transformation in bacteria?
Transformation is the process of inserting a plasmid into a bacterial cell, where the plasmid remains exogenous and floats around in the cell.
What is the role of adhesion zones in bacterial membranes?
Adhesion zones are small gaps in the membrane used for communication but are too small for plasmids to pass through naturally.
Why does the plasmid face difficulty crossing the bacterial membrane?
The phosphate backbone of the plasmid and the bacterial membrane are both negatively charged, causing repulsion.
What barriers does the intracellular space present to plasmids?
The intracellular space is negatively charged, regulated by proton pumps, and repels the plasmid.
How does CaCl2 treatment help in transformation?
Ca2+ neutralizes the negative charges on the bacterial membrane and plasmid.
Why is cooling on ice important during transformation?
Cooling stabilizes negative charges and slows molecular movement, aiding Ca2+ in neutralizing charges.
How do heat pulses assist in plasmid uptake?
Heat pulses release lipids from the bacterial membrane, creating larger openings by fusing pores with adhesion zones, allowing plasmid entry.
What role does the osmotic gradient play in plasmid uptake?
Water flows down the osmotic gradient, helping draw the plasmid into the cell.
How does heat affect intracellular negative charge?
The heat pulse shuts down proton pumps, increasing positive hydrogen ions and reducing the negative charge.
Why is LB broth added after transformation?
LB broth provides nutrients and growth factors, maximizing bacterial metabolism.
What is the lag phase?
A phase at 37°C where the plasmid is transcribed, translated, and replicated in bacteria, ensuring antibiotic resistance and plasmid establishment.
Why is plasmid replication important?
To ensure multiple copies are available for daughter cells and for expressing the DNA insert.
What assumption is made when picking bacterial colonies?
Each colony originates from a single bacterium, meaning bacteria in the colony are genetically identical.
Why are colonies picked and grown in liquid culture?
To amplify identical bacteria containing a plasmid with the specific insert for plasmid isolation.
What is the purpose of a miniprep?
To isolate plasmid DNA from bacterial cells.
How are bacterial cells collected?
By centrifuging liquid culture to form a cell pellet and discarding the supernatant.
What does the lysis buffer do?
It contains SDS and chaotropic salts to destabilize proteins and aid nucleic acid binding to silica membranes.
Why is neutralization important?
It precipitates SDS and proteins, preventing DNA degradation and improving plasmid yield.
How is DNA bound to the silica membrane?
High salt concentration facilitates binding of RNA and DNA to the membrane during centrifugation.
Why are alcohol-based washes used?
To remove salts that can interfere with downstream enzymatic reactions.
How is plasmid DNA eluted from the membrane?
Using a low ionic strength solution like water or TE buffer, followed by centrifugation to collect plasmid DNA.
What is the difference between technical and experimental controls?
Technical controls ensure the technique is working, while experimental controls represent normal conditions for comparison.
What were the technical controls in the experiment?
Insert Plate: Looking for white colonies (contain insert).
H2O Plate: Control plate with water instead of insert; expects only blue colonies.
Uncut Plasmid Plate: Ensures E. coli can be transformed by the plasmid; expects blue colonies.
TE Buffer Plate: No plasmid; expects no colonies as no resistance gene is present.
TE Buffer on LB Plate: Ensures E. coli is alive; expects growth as no antibiotic is present.
What happens if controls show unexpected results?
The experiment’s data cannot be trusted, and no conclusions can be drawn.
What information should tables include when counting colonies?
Sample name, plate type, number of white colonies, and number of blue colonies
Why is ligation diluted?
To ensure individual colonies are visible and countable.
What determines the orientation of the insert in a plasmid?
Whether one or two restriction enzymes are used in the digestion process. Using one enzyme allows for two possible orientations; using two enzymes ensures a single orientation.
Why is the orientation of the insert important?
For correct expression of the insert, the orientation must align with the 5’-3’ direction.
How can the orientation of an insert be determined?
By performing restriction digests and analyzing the resulting band patterns on an agarose gel.
What enzymes are used in the plasmid mapping digest?
EcoRI, BamHI, and a combination of EcoRI + BamHI.
Why is agarose gel electrophoresis used?
To separate DNA fragments by size and analyze band patterns to determine insert orientation
What is the purpose of using a DNA ladder?
To compare and identify the sizes of DNA fragments.
Why is Red Safe used in gel electrophoresis?
It intercalates between DNA base pairs and fluoresces under UV light, allowing visualization of DNA bands.
What forms of uncut plasmid DNA can be seen on a gel?
Supercoiled (smallest band), nicked (single-strand cut, slowest), and linear (double-strand cut).
How do single and double digests differ?
A single digest cuts the plasmid once, while a double digest cuts it twice, producing more distinct band patterns.
How does gel percentage affect resolution?
Higher agarose concentration creates smaller pores, providing greater resolution for smaller fragments but requiring longer run times.
What should be included in gel pictures?
Labeled lanes, identified band sizes, and the band patterns used to determine insert orientation.
What is Kohler illumination?
A process for providing even illumination of the field of view, reducing artifacts by adjusting the field diaphragm under different objectives.
Why is it necessary to calibrate a pH meter each time?
To ensure accuracy, using commercially available solutions of known pH for calibration.
Why are buffers important in biological reactions?
Many biological reactions are pH-sensitive and require buffer solutions to maintain a stable pH
What is Kohler illumination?
A process for providing even illumination of the field of view, reducing artifacts by adjusting the field diaphragm under different objectives.
Why is it necessary to calibrate a pH meter each time?
To ensure accuracy, using commercially available solutions of known pH for calibration.
Why are buffers important in biological reactions?
Many biological reactions are pH-sensitive and require buffer solutions to maintain a stable pH
What does spectrophotometry measure?
The concentration of a sample by analyzing how it absorbs light.
What is absorption spectroscopy?
The use of light to probe matter by measuring how a sample absorbs light at specific wavelengths.
What is %T in spectrophotometry?
The percentage of light that gets through the sample, calculated as %T = (Pout / Pin) x 100.
How is absorbance related to concentration?
Absorbance is linearly related to concentration and is calculated using the formula A = 2 - log(%T).
Why is a maximum wavelength used in absorbance studies?
To measure absorbance at the wavelength where the molecule absorbs the most light, minimizing interference from other molecules.
What are the modes of operation for a microplate reader?
Absorbance: Works like a spectrophotometer.
Fluorescence: Detects light emitted by fluorescent tags.
Luminescence: Measures natural light emission from samples.
Why are triplicates used in spectrophotometry?
To measure precision of the instrument by averaging readings from the same sample.
What is the difference between technical and biological replicates?
Technical replicates measure the same sample multiple times for precision, while biological replicates measure distinct samples for variability.
What are colorimetric assays used for?
For relative quantification by comparing samples
What are the advantages of lab studies?
More controlled environment, useful when variables of interest are identified.
What are the limitations of lab studies?
May miss interacting variables present in nature.
What are the advantages of field studies?
More realistic for examining environmental effects on variables of interest.
What are the limitations of field studies?
Harder to control and may be difficult or impossible during certain times of the year.
What is the purpose of statistical analysis?
It determines if the treatment affected the variable of interest.
What is the Null Hypothesis in statistical hypothesis testing?
It states that the independent variable does not have an effect on the dependent variable.
What is the Alternative Hypothesis in statistical hypothesis testing?
It states that the independent variable does have an effect on the dependent variable.
What does the P-value represent in statistical analysis?
The probability that the null hypothesis is supported. A low p-value suggests the independent variable had an effect, and a high p-value suggests it did not.
What is the critical value in statistical tests?
It is the threshold value, historically set at 0.05, used to compare against the probability statistic.
When should a correlation test be used?
When both the dependent and independent variables are continuous.
How should results from a correlation test be reported?
As (Pearson correlation coefficient =, n =, P =).
What does “Multiple R” represent in regression analysis?
It is the Pearson correlation coefficient.
How should results from regression analysis be presented?
As y range (dependent variable), x range (independent variable), with Multiple R, Observance (sample size), and Significance (p-value).
What does a T-test compare?
It compares a continuous dependent variable with a categorical independent variable that has two different treatments.
How should T-test results be reported?
As (t =, df =, p =).
What is an ANOVA test used for?
To compare a continuous dependent variable with a categorical independent variable that has more than two treatments.
How should results from an ANOVA test be reported?
As (F =, df = first df, second df, P =).
What is a post-hoc test used for?
To determine which treatments are different from each other after an ANOVA.
Why is a correction applied to p-values in post-hoc tests?
To account for the multiple tests being performed, which may lead to false positives. The critical value is found by dividing 0.05 by the number of pairwise comparisons.
How are bar charts used in statistical tests like T-tests and ANOVA?
They display the data with axis labels, mean plots, error bars (standard deviation), and sometimes differentiate groups using letters.
Why is it important to make a research paper attention-grabbing?
A good title and abstract increase visibility, leading to more readers and potential funding.
What should not be included in the abstract of a research paper?
Results, statistics, or references.
What does the “Acknowledgements” section of a research paper do?
It acknowledges contributions made by others to the research.
What determines the structure of a research paper?
The structure varies depending on the journal.
What are the typical sections of a research paper?
Title, Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgements, References
What sections are included in a poster presentation?
Title, Introduction, Materials and Methods, Results, Discussion, Acknowledgements, References.
How should species be referred to in a research paper?
Use both the common and scientific name the first time, then just the common name.
What is the C-C-C structure for paragraphs?
Context (topic sentence), Content (related information), Conclusion (summarizes main idea).
What is a good approach for writing a title?
Keep it clear, interesting, and concise. Include the key result.
What should an abstract include?
A short summary with context, research question, hypothesis, methods, results (no statistics), and findings in context.
What should the introduction of a research paper contain?
References, context, objective, hypothesis, predictions, and a narrowing focus.
What should be included in the Materials and Methods section?
A detailed explanation of what was done, how the experiment can be repeated, unique equipment used, and reasons for excluding data.
What is the purpose of the Results section?
To discuss the findings, including statistical tests and results, sample size, and a narrative. Present results as tables or figures.
What should be avoided in the Results section?
Interpreting biological mechanisms.
What should the Discussion section address?
Interpretation of results, biological explanations, comparison with other studies, and the importance of the results.
What should be included in the Acknowledgements section?
A list of people who supported the research.
How should references be formatted in a research paper?
References should be in alphabetical order, and in-text citations should avoid repetition by citing once and referring back to it.
What is important when writing the Materials and Methods section?
It should contain enough detail to allow the experiment to be repeated by others, including any necessary equipment and methods.
What is the difference between primary and secondary scientific sources?
Primary sources provide direct evidence about an event or object, usually including methods and results. Secondary sources describe, analyze, or comment on a primary source, often published in a book or journal.
What is a meta-analysis?
A meta-analysis combines several empirical studies to identify an overall pattern in results. It includes methods describing how articles were selected and results with a graph showing effect size. Larger effect sizes indicate a bigger difference between treatments.
What characterizes an experiment in scientific communication?
An experiment manipulates one or more variables to observe the effects of a treatment, usually following a full factorial design.
What is an observation in scientific research?
An observation uses natural variation to study the effects of the environment on subjects, offering a way to test hypotheses.
What is the purpose of a model in scientific research?
A model allows scientists to test their understanding of a system by manipulating important variables to predict how the system behaves.
What is a review article?
A review summarizes existing knowledge on a topic, identifies gaps, and may propose new hypotheses for further testing.
What role do blogs and news stories play in scientific communication?
Blogs and news stories provide a way to quickly comment on or disseminate research to the public.
How do books differ when written for a scientific audience?
Books written for a scientific audience are more rigorous and correct, with higher standards of evidence and clarity.
What is the typical sequence in a research method?
Observation, Question, Hypothesis, Prediction, Test, Report Results, Recommend Action.
What is the peer review process in scientific publishing?
A scientist submits a paper, which is reviewed by editors and scientists. If revisions are suggested, the paper is returned to the author. If reviewers give positive feedback, the editor recommends publication.
What happens if a paper is rejected during the peer review process?
The scientist addresses the reviewers’ suggestions and submits the revised paper to another journal.
How do different journals affect reference styles?
Different journals have different reference styles. It’s important to pay attention to in-text citations and the format of references.
