Five Ways to Improve Your Transformation Experiments

Transformation is the process through which bacteria can pick up foreign DNA from the surrounding environment. The newly acquired genetic information is both stable and heritable.

NEWS_2.27.15_Five_Ways_Thumb.jpgIn the laboratory, we can transform bacteria with small, circular pieces of DNA called plasmids. These plasmids are engineered to contain genes from different sources. Once transformed into bacteria, the plasmids turn the bacteria into living factories to create medications, vitamins and other useful products. For example, scientists use bacteria to make insulin, the medication used to treat diabetes. In the classroom, we can program E. coli with a jellyfish gene, making them glow bright green! Trust me, this experiment will amaze your students.

Our transformation experiments have been optimized these experiments to be robust and reproducible in your classroom laboratory. Here are five guidelines to ensure your students have experimental success.

  1. Preparation of the source plate: For best results, the source plate needs to be fresh! This makes sure the cells are healthy and ready for transformation. Be sure to streak out the BactoBeads™ on an agar plate 16-20 hours before the laboratory period. Do not store the plate in the refrigerator before use.
  2. Resuspension of the bacteria: After the bacterial cells are added to ice-cold calcium chloride, two things can go wrong. One problem is that the cells are not completely resuspended. This means the plasmid will not come into contact with most of the bacterial cells. The second problem is the cells get too warm during the resuspension. This affects the temperature differential essential for the heat shock (see #4). We recommend resuspending the cells by pipetting up and down until no clumps are visible. In addition, to avoid warming the cells with your hands, hold the top of the tube instead of the bottom where the cells are.
  3. Amount of plasmid DNA: Understandably, too little plasmid DNA will reduce the transformation efficiency, but did you know that adding too much plasmid could also affect your results? Although the plasmid concentration in our experiments has been optimized, pipetting error can affect your student’s results. Make sure your students know how to accurately pipet before the experiment by practicing with kit S-44, Micropipetting Basics. (LINK to S-44)
  4. Temperature: It is believed the combination of chemical ions and the rapid change in temperature (i.e. the “heat shock”) alters the permeability of the bacterial cell wall and membrane. This allows the DNA molecules to enter the cell. For best results, be sure to keep the cell suspension ice-cold before and after the heat shock. Additionally, before heat shocking, use a thermometer to confirm that the water bath reaches at 42°C.
  5. Recovery period: During this incubation, the bacteria repair their cell walls and express the antibiotic resistance gene. Be sure to allow the cells enough time to recover. If the cells are plated too soon, they may not be able to grow on the selective media. However, after 30 minutes of recovery, the bacteria will double, meaning that many of the “transformants” are produced by cell division and not by transformation.

For more information about troubleshooting your transformation, be sure to check out the information on our Resources Page.

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The power of positivity in human language

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Take a moment to think back to the past — to the events of last week, last year, or even back to your childhood. Are you more likely to describe these memories in positive or negative terms? For most people, the tendency is to fondly remember past events and to discuss things using positive language. Interestingly, this bias has been found in people across the world, bridging both culture and language. Psychologists have referred to this phenomenon as the “Pollyanna principle,” the inclination to describe things in a positive light, even when they are negative.

A recent study took an in-depth look into this bias within language, taking advantage of some of the internet resources we use everyday – Twitter, Google Books, and online newspapers. The researchers started by selecting 10 languages from around the world. Next, they collected datasets of approximately 10,000 words for each language, using computer programs to pull common words from the online resources. Finally, native speakers for each language were paid to rate the words on a scale from 1 to 9 based on how positive or negative each word made them feel.

Try it yourself on this random list of common English words, thinking about how positive or negative each word is on a 9-point scale. You can scroll down to find the results from the study.news_4.1.15_power_of_positivity_table1-1.jpg

 

The results of this analysis were shocking; every language tested showed a positive bias in their word scores, regardless of what source was used to collect the words. This means that overall, language tends to include more frequently used positive words than negative words! Spanish was found to be the most positive of the languages tested, but even the lowest ranked languages still had more positive than negative words.

While it’s impossible to determine the reasons for the bias using this study alone, it does shed some light on how communication effects happiness. Scientists still do not know if language is responsible for the Pollyanna principle, or if our natural positivity might have influenced language. In the meantime, if you’re looking for a little extra happiness in your life it might be worth taking a Spanish language class!

The original article is available for open access by the Proceedings of the National Academy of Sciences at http://www.pnas.org/content/112/8/2389

For more fun with language happiness, including analysis of books, movies, and twitter check out the Hedonometer at: http://www.Hedonometer.org

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Exploring our Chromosomal DNA

NEWS_1.19.15_ Chromatin_Structures_Banner.jpgThe nucleus is a dense organelle that contains the genetic material of a cell. How dense? The human genome is around 3 billion nucleotides and the average length of a nucleotide is 0.6 nanometers, so stretched out the DNA in a cell would be around 1.8 meters or 5 ft. (Here’s the math: 3.0 × 109 x 6.0 × 10-10 meters = 1.8 meters). Cells must package 5 feet of genetic material into a nucleus of around 6 micrometers. They do this by tightly coiling the DNA around histone molecules to form chromatin. Chromatin is then coiled and looped and coiled and looped some more as illustrated above.

How many loops? That’s one question that a group of scientists recently tackled with a landmark study that begins to map how the human genome folds itself inside the nucleus of a cell. The answer – around 10,000 – is much lower than had previously been predicted.  The study also found that most loops are relatively small, less than 2 million base pairs. This work adds support and detail to the hypothesis that DNA loops play a crucial role in gene regulation. Loops often bring together distant enhancers and promoters that dictate gene expression.  These are sometime referred to as secret switches. As loops form and un-form, different “secret switches” are turned on and off.

The 3D map of nuclear DNA folding and looping in on itself was created using some fundamental tools of the trade.  First the cellular chromatin structure was treated with formaldehyde to preserve its 3D structure. Next, restriction enzymes cut the DNA into tiny pieces and biotin was added to mark the newly cut ends. Neighboring ends were then ligated together. These ligation junctions were then sequenced. For more on restriction enzymes check out this previous blog or one of our several kits that introduces students to this technology (for example, DNA Fingerprinting Using Restriction Enzymes).

S.S.P. Rao et al., “A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping,” Cell, doi:10.1016/j.cell.2014.11.021, 2014.

Mutant Plants to the Rescue!

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Bioremediation is a management tool that addresses environmental pollution. In bioremediation organisms remove or neutralize pollutants from a contaminated site. Often these organisms are already present at the site.  For example a group of scientists from Rutgers University discovered a strain of bacteria that can breathe either oxygen or uranium in the soil of an old ore mill in Rifle Colorado. Other times organisms adept at containing or converting the specific pollutant are introduced to the site. In such cases the use of native species is encouraged in order to maintain the integrity of the local ecosystem.  

One area of biotechnology research involves finding organisms that are optimally suited for the clean up of a certain chemical and understanding what adaptations enable this service. An example of such work can be found in a Science article that received lots of attention because of its explosive subject matter – the bioremediation of TNT by the plant species Arabidopsis. 

NEWS_10.13.15_Arabidopsis_Fig1Arabidopsis, or more specifically Arabidopsis thaliana, is one of the most common plant model organisms. One advantage of Arabidopsis’ popularity is that its genetic makeup has been well studied and documented. Additionally, there are many existing mutant Arabidopsis lines, allowing scientists to rapidly screen for useful adaptations.

The TNT study began with planting several different mutant lines of Arabidopsis thaliana in both uncontaminated and TNT treated soil and looking for individuals with enhanced TNT tolerance. This is exactly what the scientists found – a line of plants that had significantly more root growth in the TNT soil than the other lines but the same amount of growth in the uncontaminated soil.

The next step was to discover the genetic underpinning of this trait. The mutation turned out to be a single nucleotide deletion in a gene called MDHAR6. This was a surprise to the scientists who were expecting to find a new or over-expressed detoxifying enzyme. MDHAR genes code for the antioxidant enzyme monodehydroascorbate reductase which reduces oxidative stress in plants by helping to recycle vitamin C. How could a loss of function mutation in this gene allow plants to tolerate TNT? 

To investigate this question scientist expressed MDHAR6 in E. coli and then purified the resulting protein by affinity chromatography. Experimenting with this protein product they found that the enzyme caused a one electron reduction in TNT. This reaction used NADH and created reactive oxygen species (ROS) that at high levels can damage cell structures. 

Returning to the plants, the scientists measured oxidative stress in both mutated and wild-type plants planted in TNT and found higher levels of stress in the normal plants. Finally, they transformed the MDHA6 gene back into the mutated line. This resulted in restored TNT toxicity and confirmed that plants deficient in MDHAR6 are more tolerant to TNT.

Arabidopsis is too small to efficiently remove TNT from polluted sites. However, the MDHAR6 gene can be disrupted or “knocked out” in other plants using traditional breeding methods. This is good news for thousands of sites that have been contaminated with residual TNT following explosions. It’s also possible that this strategy might lead to novel bioremediation strategies for other contaminants. 

To explore bioremediation in your classroom, we recommend starting with the “Bioremediation by Oil Eating Bacteria” Kit.  You can also test the effects of environmental pollution on plants with our “The Dose Makes the Poison” kit