STEM is taught by Dr. Kevin Crowthers. In this class, we navigate the world of research by using online resources and learning how to read scientific articles. We use this knowledge not to be tested on it, but to perform and log our own experiments and write scientific articles. One of the projects we completed was the Build Something Project, which introduced us to the engineering process. We are currently completing a five-month independent research project! I am working with E. coli and algae C. reinhardtii and am studying their movement in the BSL-2 lab at school. Working at a lab and planning a five-month long project sounded overwhelming at first, but eventually with the guidance of Dr. Crowthers and my peers the scientific process came naturally! Stay tuned for a more detailed description of my STEM project!
Exposing biohybrids composed of Escherichia coli and Chlamydomonas reinhardtii to multiple attractants- d-galactose, bicarbonate, and a light source, results in a directional, biased taxis towards these attractants.
Many medications, including chemotherapeutic drugs, are either taken orally or through intravenous therapy (IV). However, these drugs are often designed to be taken over a long period of time and cannot be transported directly to the target site, thereby reducing the drug’s efficacy. Some studies have used microorganisms as “engines” for cargo-carrying nanovehicles in a targeted drug delivery system because of their ability to respond to environmental stimuli. Currently, these nanovehicles utilize a magnetic field to achieve biohybrid mobility, but they are unfeasible biomedically. To avoid the use of electromagnetics, this project proposes a design that uses the flagellar chemotactic movement of E. coli towards d-galactose, an isomer of glucose, and the phototaxis of microalgae C. reinhardtii towards light as a steering mechanism. The biohybrids were created by coating E. coli cells with four layers of polyelectrolytes to bind C. reinhardtii cells to the cell surface. The biohybrids were placed in a cell migration assay to test the effects of both a light source and chemoattractant on the directionality and drift velocity of the biohybrid. It was found that the drift velocity of the E. coli/ C. reinhardtii hybrid was higher than the drift velocity of a singular E. coli cell or C. reinhardtii cell. Qualitative analysis suggested that the presence of multiple attractants results in a more directional nano-vehicle because more biohybrids migrated to an area that contained more than one attractant. This design can be applied to targeted drug delivery systems in which a light source is inserted near a target site to attract the biohybrid and reduce the drug’s exposure to healthy cells, thereby reducing the chances of a patient experiencing side effects.
To what extent is the movement of Escherichia coli and Chlamydomonas reinhardtii hybrids optimized by both a light source and chemical attractants?
If C. reinhardtii is used to form a biohybrid structure which is currently only motile through the natural movement of E.coli, the biohybrid will have a more directed path if both organisms are moving through phototaxis and chemotaxis as a transport mechanism than if a single organism is only using one transport mechanism.
While chemotherapy is one of the most common forms of cancer treatment, it and similar medications often result in adverse effects including hypertension, fatigue, and headaches. 85% of chemotherapy patients report feeling fatigued after a session. Other effects of chemotherapy such as hair loss occur when healthy cells are exposed to certain medications. This is because the drugs are not targeted towards one site, which is especially harmful in therapeutics that are only designed to affect a certain area or type of cell.
Scientists have devised methods to limit contact between therapeutics and healthy cells by exploring methods of targeted drug delivery, and microorganisms such as bacteira and microalgae have been considered as nanovehicle "engines" because of their natural flagellar movement.
However, this flagellar movement is random and oscillating, which can be harmful in a system meant to carry drugs toxic to healthy cells. In order to combat this and achieve a more targeted nano-vehicle, a property of microorganisms called “taxis”, in which a microorganism is able to respond to external stimuli (thus inciting biased movement) is being studied for its application in targeted drug delivery.
The biohybrids were created by coating E. coli cells with polyelectrolytes Poly(allylamine) solution (Sigma Alrich, 10%, mw 56,000 Da) and Poly(sodium-4 styrenesulfonate) (Sigma Alrich, 30%, mw 65,000 Da) solution, both diluted to 1mg^-1mL in 100mM salt solution. Around 10 colonies of E. coli were placed in a microcentrifuge tube and washed once with NaCl. Four layers of polyelectrolytes were absorbed onto the cells. Each coating step of 2mL of polyelectrolyte solution lasted for 10 minutes and was followed by centrifugation at 12000g for 5-7 minutes. Between each coating step, cells were washed with NaCl and centrifuged at 12000g for 5 minutes. After the cells were coated with the polyelectrolytes, the cells were coated once with 2mL of C. reinhardtii solution.
The biohybrids were placed at the end of a 5mL plate with a channel cut out of the end. 15 µL D-galactose and L-arabinose (diluted to 20%) were added to the end of the channel and left to diffuse for 20 minutes. After this period, the channel was filled with water and placed in a dark area with a light source pointing through a slit in the same direction of the sugars. Biohybrids taken using a loop were placed at the other end of the channel. The movement of the biohybrids was photographed and measured at the end of a 24 hour period.
The mean distance traveled by the particles in each of the biohybrid assays was calculated. This was compared with the mean distance travelled by C. reinhardtii to light or bicarbonate, and E. coli to d-galactose.
The results of the biohybrid migration assay to d-galactose, bicarbonate, and a light source were collected using either a USB microscope or an iPhone camera. The pictures were cropped until each end of the plate fit the frame, and the pictures were lined against a digital ruler. This allowed for a uniform measurement of the distances travelled by each particle. The results of the biohybrid assays are summarized in the table below.
The table shows the maximum distances traveled by the biohybrid, E. coli, and C. reinhardtii particles in each trial. The figures show the comparison of the biohybrid's path with the other particles.
A Wilcoxon Rank-Sum Test was performed on this data. This type of statistical test was used because of the small sample size and inconsistency of the data, which made it unfeasible to be tested with a t-test, which requires a normal data set. The results of this statistical test were recorded in the table below:
The objective of this project was to engineer a biohybrid microswimmer, or a structure composed of both living organisms and biomaterials, that was proficient in both chemotactic movement and phototactic movement when exposed to bicarbonate, d-galactose, and a light source. This objective was achieved by coating E. coli cells with polyelectrolytes that bound it to C. reinhardtii, a type of microalga that can achieve phototaxis. The second objective of this project was to engineer a biohybrid that could surpass the speed of individual E. coli or C. reinhardtii cells- that is, travel a farther distance than the single cells after a 24 hour time period. After calculating the maximum distance that each microswimmer traveled after a 24 hour time period within a channel cut into a 5mL plate, it was found that the biohybrids (which were exposed to a bicarbonate, d-galactose, and a light source) performed better, on average, than E. coli did when moving towards d-galactose and than C. reinhardtii did when moving towards bicarbonate. However, the biohybrids failed to travel a farther distance than C. reinhardtii did when exposed to a light source. Furthermore, a statistical analysis test (Wilcoxon Rank-Sum Test) done on the data suggested that the biohybrids did not perform better than the control experiments, and that the results were insignificant.
In order to improve the results of these tests, a larger sample size of data will be needed to achieve a more consistent and normal data set. In the future, different processes of engineering these biohybrids will need to be completed to gain more significant results. However, the results of these studies suggest that the biohybrids do exhibit a biased movement towards chemical and light attractants. This study takes a step into the world of microorganism behavior and their applications in real life. In a biomedical context, this type of technology could be used to achieve targeted drug delivery, a process that could reduce the risk of side effects and greatly reduce infusion time of long-term therapeutics without the use of external mechanisms to guide the cargo carriers. Biohybrid microswimmers explore a link between human achievement and the existing laws and behavior of creatures in nature.