Biology 336: Suggestions For Independent Molecular Biology Projects.
The following are a list of experiments that are to be done independently during the last 5 weeks of the semester. Students can work as groups of 2-3. By the sixth week, students should choose one of the experiments and notify the instructor. By the end of the eighth week, students should submit a planned protocol and schedule to meet with the instructor to insure it will be successful. By the beginning of the eleventh week, students should be ready to start the project. The project should be done during the scheduled lab hours but can also be done at different times with permission of the instructor. During the last week of the semester, student groups should appoint a speaker to present their results to the class using visual aids. A 3-5 page scientific paper (with abstract, introduction, materials and methods, results, and discussion) is then to be submitted on the day the class final is given. Students groups will also present their results at a poster session held each semester by Baldwin-Wallace College.
If any student has a project idea other than those listed below, see the instructor. Such original projects are encouraged, however, we must work within the time and budget constraints of the class.
The Experiments
Measuring the effects of selection
In this project, students will empirically establish the role selection plays in molecular biology experiments. The project will involve two experiments:
a. Students will make competent E. coli and transform them with pUC18 plasmid. Bacteria will be grown on plates containing LB media/X-gal with and without ampicillin. The number of colonies grown in each case, along with the % blue will be determined to demonstrate the significance of selection with ampicillin.
b. Students will begin with a yeast strain that contains two plasmids, each with a different selectable marker. One plasmid contains URA and the other contains TRP. Yeast will be grown in media that contains tryptophan, thus there is no selective pressure to maintain this plasmid. Students will take aliquots of cells over time and plate. They will then replica-plate the colonies on media lacking both uracil and tryptophan to determine the degree of plasmid loss over time.
Skills: Pouring plates, making competent bacteria, transforming bacteria, plating cells, and replica-plating.
Biochemical analysis of a restriction enzyme
In this project, students will determine if the recommended reaction conditions for restriction enzymes are optimal. Students will obtain a restriction enzyme (EcoR1 and HindIII) and determines its pH and temperature profile. This will be done by cutting lambda DNA at different temperatures and comparing the extent of digestion on an agarose gel. Students will also make restriction enzyme buffer and vary the pH and repeat the same experiment.
Skills: Restriction digests, pouring agarose gels and electrophoresis, making buffers.
Measuring the activity of alkaline phosphatase
In this project, students will be empirically demonstrating the utility of alkaline phosphate in cloning experiments. Students will cut the pUC18 plasmid with HindIII. Half of the vector will be treated with alkaline phosphatase and the other half will remain untreated. Students will the cut lambda DNA with HindIII and isolate the 2.2 kb fragment. Ligation reactions with both the alkaline phosphatase-treated and untreated cut-pUC18 will then be set up. The number of colonies and % blue will be determined.
Skills: Restriction digests, alkaline phosphatase digests, pouring agarose gels and isolation of DNA from the gels, setting up restriction digests, transformations.
Measuring the selection coefficient of yeast expressing Xenopus TFIIIA
Expressing Xenopus TFIIIA in yeast is costly, as yeast do not use this protein, and yet expend energy synthesizing it. In this experiment, students will determine if yeast expressing TFIIIA have a selective advantage over those not expressing this frog protein. Students will inoculate a culture with equivalent amounts of yeast that do and do not express TFIIIA. After time intervals, students will plate the yeast and replica-plate on media containing X-gal. The % blue will be determined. The cultures may be modified to contain varying amounts of nutrients.
Skills: Growing and setting up cultures, plating, and replica-plating.
Testing Internet Procedures:
The internet houses many lab procedures that may be different from what we learned in lab. Students will find and download some of the procedures and judge their effectiveness.
Measuring plasmid loss in bacteria
Plasmids can be spontaneously lost by bacteria. Students will attempt to measure the incidence of this loss using a plasmid that expresses green fluorescent protein (GFP). Bacteria will be transformed with GFP. Two cultures with transformed bacteria will be set up: one with ampicillin to select plasmid and one without. Aliquots from both cultures will be taken at various time intervels and % green coloinies will be determined (loss of green indicates loss of plasmid).
Skills: Bacterial transformations, setting up cultures, and plating.
Measuring the mutagenic effects of ethidium bromide.
In this project, students will determine to what extent ethidium bromide is mutagenic. Yeast containing both pG1-803 and pY25SR will be grown in media with varying amounts of ethidium bromide (along with a control culture without ethidium bromide). The yeast will then be plated and replica-plated. Both the number of colonies and the %blue will be scored to estimate the frequency of mutagenesis.
Skills: Growing and setting up cultures, plating, and replica-plating
Measuring the mutagenic effects of UV light
In this project, students will determine to what extent UV light is mutagenic. Yeast containing both pG1-803 and pY25SR will be grown in media and plated. Two plates will be made and one plate will be exposed to UVc light. Both plates will be grown and replica-plated. Both the number of colonies and the %blue will be scored to estimate the frequency of mutagenesis.
Skills: Growing and setting up cultures, plating, and replica-plating
Sub-cloning experiment
In this project, students will begin with a pG1-803 plasmid that contains a mutant version of TFIIIA containing more than one point mutation. Students will use standard sub-cloning procedures (cutting with restriction enzymes and pasting with ligase) to separate the two mutations.
Skills: Large scale plasmid prep from bacteria, restriction digests, pouring gels and isolating DNA from gels, setting up ligation reactions and transforming bacteria and small-scale plasmid preps.
Continuing to screen for TFIIIA mutants
In this project, students will continue with the techniques learned in the first 10 weeks and screen for TFIIIA mutants. Specifically, students will use the Western blot to continue screening whites isolated by the class. Plasmid DNA will be isolated from those yeast that pass this screen and used to transform bacteria. Plasmid DNA will be isolated from the bacteria and then used to transform the yeast to insure a white phenotype. Other variants on this screen may also be available depending on what is isolated by the class.
Skills: SDS PAGE gels, Western blotting, plasmid isolation from yeast and bacteria, transformation of yeast and bacteria.
Measuring effect of plasmid size on transformation efficiency
In this experiment, students will determine what effect (if any) the size of a plasmid has on transformation. Students will prep two different plasmids - a small one containing green fluorescent protein (GFP) and a large one without (pg1-803). DNA concentrations will be determined and competent bacteria will be transformed with each in isolation, along with an
equal concentration of both. Number of colonies and % green will be determined. Alternatively, students will prep plasmids of three differing sizes and compare the tranformation efficiency using the same amount of the three different plasmids.
Skills: large scale DNA preps, DNA concentration determinations, making competent
Exploring bacterial transformation conditions
In this experiment, students will explore some of the conditions employed in standard bacterial transformations. Calcium chloride was used in class (100 mM) to make bacteria competent. What if the concentration is altered? What if another salt is used? Would the use of carrier DNA improve bacterial transformations? Can the 45 minute ice- incubation step be extended or decreased?
Skills: Making solutions, making competent bacteria, bacterial
Observing nuclear localization
Some proteins are localized to the nucleus. To observe this phenomena, students will transforms yeast with plasmids containing a nuclear protein fused to GFP and plasmids expressing GFP alone. Students will then take photographs of both strains. Some aspects of this experiment may need ironing out.
Skills: Pour plates, transform bacteria and yeast, microscopy.
Using PCR to Compare ribosomal RNA from Different Organisms
The 16S ribosomal RNA from various organisms will be compared by PCR analysis. Students will be given genomic DNA from cow, pig, chicken, and E.coli, but should also isolate genomic DNA from themselves and another organism. PCR will then be run and the results will be analyzed.
Skills: Genomic DNA isolation, PCR, and gel electrophoresis