In this kit, students will develop skills in gRNA design and will be able to simulate CRISPR-Cas9 technology to modify bacterial DNA. Given the vast differences in climate between Mars and Earth, bacteria that can easily thrive on Earth struggle to survive on Mars. Therefore, students will need to engineer bacteria, by editing its DNA so that it can survive, thrive, and help terraform Mars.
Terraforming is a theoretical process that helps create a habitable environment that mimics conditions on Earth, allowing humans to colonize planets and live life similarly to how they would on Earth. Many issues are currently threatening human life on Earth, like overcrowding and the climate crisis, which has led to investigating potential habitable planets for terraforming. It is a difficult task to find an alternative habitable planet for the human race, but there has been much discussion about the great potential in terraforming Mars. Mars is highly similar to Earth in many ways including: sun rotation, axis of rotation, and having distinct seasons. While it has all elements that are essential for human life like: water, carbon dioxide, and nitrogen, the composition of the atmosphere is not ideal. The atmosphere is mainly composed of carbon dioxide, and is 100 times thinner than the Earth’s atmosphere. A low density atmosphere makes it quite difficult for the planet to retain heat, making the climate very cold and dry.
During the terraforming process, a major goal will be to thicken the atmosphere and to increase the overall temperature by releasing the greenhouse gases from Mars. Once the ideal temperature is reached, enough oxygen needs to be supplied to the planet to sustain human life. It is believed that this can be achieved by utilizing synthetic biology, where organisms are redesigned to display unique and useful characteristics.
In this experiment, students will be looking at how a CRISPR-edited extremophile gene affects the capability of a bacteria to grow on Earth versus Mars. The extremophile gene (ext gene) is derived from cyanobacteria, one of the oldest organisms on Earth. It is thought to be one of the main contributors of oxygen, that makes up over 20% of the Earth’s atmosphere. Cyanobacteria are extremophiles that are capable of converting carbon dioxide into organic molecules, like oxygen, through carbon fixation. Extremophiles have the ability to grow and flourish in hostile environments, like Mars’ extreme cold and dry climate.
In this simulated experiment, students will use CRISPR-Cas9 to introduce the ext gene to E. coli DNA, a common bacterial strain on Earth that thrives in temperate climates. In the first module, students will design guide RNAs (gRNAs), an important aspect of the CRISPR-Cas9 system. These gRNAs are short and synthetic RNA sequences that are complementary to targeted pieces of DNA, and are capable of signaling cas proteins to create complexes with the target DNA. Within the E. coli genome, a sequence with matching flanking regions to the ext gene from cyanobacteria was identified. This sequence will be the target DNA sequence for the CRISPR-Cas9 system. In the second module, students will introduce the ext gene to a fluorescently tagged Cas9 plasmid and complete a ligation. The ext gene will be introduced into the target sequence through homologous direct repair (HDR). In the third module, a transformation procedure will be used, where competent cells will be able to uptake the CRISPR plasmid, which contains the newly incorporated ext gene. Transformed cells will be plated on both Mars and Earth plates. Here, students will be able to determine how the addition of the ext gene changes the bacteria’s growth on each planet’s surface.
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