MIT Class Site
Laura Maria Gonzalez
May 21, 2021
The ultraviolet index (UV index) is an international standard measurement of the strength of sunburn-producing ultraviolet (UV) radiation. It was
developed in 1992 and standardized by the World Health Organization and World Meteorological Organization in 1994. The purpose of the index is to
help people protect themselves from UV radiation which can cause sunburn, skin aging, DNA damage, and skin cancer. Sunburn to the skin is related
to wavelength, the shorter the wavelength the more damaging.

The UV index can be calculated from a direct measurement of the UV spectral power at a given location. In weather reports it is usually predicted
based on a computer model and represents the UV intensity around the sun's highest point in the day. The computer model takes into account the
sun elevation and distance, stratospheric ozone, cloud conditions, air pollutants, surface albedo, and ground altitude.
The project was inspired by Coral that have a fluorescent pigment sunblock. Corals live in symbiosis with zooxanthella. The zooxanthella
photosynthesize and produce food for the coral, but the coral needs a defense mechanism against exposure to too much UV light. They protect
themselves while providing for their partner by absorbing Ultraviolet Radiation (UVR) and reflecting photosynthetically active radiation (PAR). They
are able to do this through florescent pigments that absorb damaging wavelengths of light and emit it as pink or purple.
Coral fluorescents, Great Barrier Reef Foundation
Acropora Millepora, Reef Builders
While initially starting with UV sensing, bacterial shading makes use of next generation synthesis techniques to create a blue light sensing genetic
circuit that can express a variety of color pigmentation in E.coli. The project also leverages the advantages of microfluidics/millifluidics to create a
compact chemostat that can provide bacteria with constant nutrition and oxygen to ensure cell growth for days to weeks. Bioproduction of the cells
will also be evaluated to understand the effects of media, temperature, and sugar on pigmentation density.
Transform E. coli cells with a photoreceptor that expresses a chromoprotein

For bacteria to react to light it must first be engineered to include a photoreceptive system. The goal of Bacterial Shading is to design a one plasmid,
light sensing, two component system (TCS) consisting of a transmembrane histidine kinase and a response regulator that controls a genetic circuit
for the expression of chromoproteins (figure 1). While TCSs are often also two plasmid systems, the bacterial shading design will incorporate the one
plasmid TCS pDawn to express a multitude of variants of the chromoprotein amilCP in a cost effective manner.
Most TCS are also two plasmid systems. For simplicity and cost reduction, Bacterial Shading implements the YF1/FixJ TCS. This optogenetic system
is a TCS, but is contained within a singular plasmid developed by Ohlendorf, Robert, et al. The system, named pDawn, is activated with blue light at
470nm. Under blue light the YF1 receptor phosphorylates a FixJ response regulator that activates the pFixK2 promoter. pFixK2 then promotes the
expression of the cI repressor which represses the promoter pR thereby activating the gene in the multiple cloning site (MCS). For Bacterial Shading,
amilCP was chosen as a reporter.

The first step towards the transformation of E.Coli cells with a YF1/FixJ TCS is to design the plasmid. Two plasmids were used to construct the design
- the pDawn plasmid and the mUAV plasmid containing the amilCP gene. (Figure 2). The YF1/FixJ system from the pDawn plasmid is first cloned into a
pET-21(+) vector via Twist Bioscience. Restriction cloning was then selected as the method to insert amilCP into the MCS of the newly constructed YF1/
FixJ plasmid.
Develop a millifluidic chemostat for the photoreceptive E.coli cells

Through a millifluidic chemostat, E.coli can be kept alive within an enclosed system. The proposed device is able achieve this through the control of
inputs such as media and oxygen as well as the output of waste. By operating at the millifluidic scale, the device can also keep the bacteria alive for
weeks at a time while using less resources. The proposed research also investigates fabrication methods that limit the amounts and complexity of
PDMS control layers typically deployed in microfluidics through the use of a milled polycarbonate design.
Integrate into existing building infrastructure

The objective of bacterial shading is to be integrated within IGU assemblies for their incorporation into buildings. To make this cost effective the
system should be able to tie into existing building infrastructure such as greywater systems and heating, ventilation, and air conditioning (HVAC)
systems. Further editing of the bacteria from heterotrophs to autotrophs is also proposed as a method of carbon sequestration to provide additional
value and use possibilities.
Hamilton, Dr. Ian, Dr. Harry Kennard, Oliver Rapf, Dr. Judit Kockat, and Dr. Sheikh Zuhaib. “2020 Global Status Report for Buildings and Construction.”
Global Alliance for Buildings and Construction. UN Environment Programme, 2020. Web. 2021.

Levskaya, Anselm, et al. “Engineering Escherichia coli to see light.” Nature 438.7067 (2005): 441-442.

Liu, Zedao, et al. “Programming bacteria with light—sensors and applications in synthetic biology.” Frontiers in microbiology 9 (2018): 2692.

Ohlendorf, Robert, et al. “From dusk till dawn: one-plasmid systems for light-regulated gene expression.” Journal of molecular biology 416.4 (2012): 534-542.

Fernandez-Rodriguez, Jesus, et al. “Engineering RGB color vision into Escherichia coli.” Nature chemical biology 13.7 (2017): 706-708.

Liljeruhm, Josefine, et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering 12.1
(2018): 1-10.

Lee, Kevin S., et al. “Microfluidic chemostat and turbidostat with flow rate, oxygen, and temperature control for dynamic continuous culture.” Lab on a
Chip 11.10 (2011): 1730-1739.

Groisman, Alex, et al. “A microfluidic chemostat for experiments with bacterial and yeast cells.” Nature methods 2.9 (2005): 685-689.

Long, Zhicheng, et al. “Microfluidic chemostat for measuring single cell dynamics in bacteria.” Lab on a Chip 13.5 (2013): 947-954.

Walsh, Matthew E., et al. “3D-Printable Materials for Microbial Liquid Culture.” 3D Printing and Additive Manufacturing 3.2 (2016): 113-118.

Keating, Steven J., et al. “3D printed multimaterial microfluidic valve.” PloS one 11.8 (2016): e0160624.

Patrick, William G., et al. “DNA assembly in 3D printed fluidics.” PloS one 10.12 (2015): e0143636.

Unger, Marc A., et al. “Monolithic microfabricated valves and pumps by multilayer soft lithography.” Science 288.5463 (2000): 113-116.

Ching, Terry, et al. “Fabrication of integrated microfluidic devices by direct ink writing (DIW) 3D printing.” Sensors and Actuators B: Chemical 297
(2019): 126609.

Melin, Jessica, and Stephen R. Quake. “Microfluidic large-scale integration: the evolution of design rules for biological automation.” Annu. Rev.
Biophys. Biomol. Struct. 36 (2007): 213-231.

Bader, Christoph, et al. “Grown, printed, and biologically augmented: An additively manufactured microfluidic wearable, functionally templated for
synthetic microbes.” 3D Printing and Additive Manufacturing 3.2 (2016): 79-89.

Craig, Salmaan, and Jonathan Grinham. “Breathing walls: The design of porous materials for heat exchange and decentralized ventilation.” Energy
and buildings 149 (2017): 246-259.

Elrayies, Ghada Mohammad. “Microalgae: prospects for greener future buildings.” Renewable and Sustainable Energy Reviews 81 (2018): 1175-1191.

Callaway, E. “Carbon Dioxide-Eating Bacteria Offer Hope for Green Production.” Nature 576.7785 (2019): 19-20.

Smith, Rachel Soo Hoo, et al. “Hybrid living materials: digital design and fabrication of 3D multimaterial structures with programmable biohybrid
surfaces.” Advanced Functional Materials 30.7 (2020): 1907401.

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Growth Chamber
Pneumatic Channels
Output Port
Media Channel
Input Port
The objective of a millifluidic chemostat is to keep E.coli alive within an enclosed system for days to weeks at a time. The design took into account
three main considerations. The first was to use millifluidic volumes in order to cover large amounts of surface area while minimizing required media
usage. The design also took into account the continuous supply of media and oxygen and removal of waste necessary to maintain cell viability for
long durations. Lastly, the millifluidic chemostat fabrication needed to be scalable so that the process could be used to manufacture larger
modules on the scale of several tens of cm in width and length. This means limiting the amounts of PDMS needed for actuation control and using
milling as the main fabrication method.

The millifluidic chemostat prototype will be 90mm in width by 82mm in length by 4mm in height and consist of two main features: a growth chamber
and a media channel based on a design by Lee, Kevin S., et al. The growth chambers will contain the E.coli cells and are connected to input and
output ports via the media channel. The cells can be passed nutrient and oxygen through a system of pneumatic channels that control three valves
and actuate mixing in the growth chamber through three additional channels.
The device has four main operational stages. The first is inoculation. In this stage the output valve is open along with its adjacent chamber valve to
allow for the insertion of cells into the growth chamber. The input port isn’t used in this stage to prevent any contamination of incoming media. The
second operational stage is media loading. All valves are except the input our closed to full load the media channel. This phase is followed by the
mixing stage where the valve adjacent to the input port is opened and media is allowed to enter the growth chamber through pneumatic actuation
by alternating positive and negative pressure in the various chambers in a clockwise direction. The final stage is waste removal. This is
accomplished by closing all ports except the output and output adjacent valve and applying positive pressure throughout the growth chambers to
push the waste out.
The experimental setup and testing of the millifluidic chemostat will follow a similar structure to that presented in Keating, Steven J., et al. The
millifluidic chip input will be connected to media and compressed air inputs. Both inputs would be pneumatically controlled with regulators
connected to a microcontroller to ensure stable conditions during long durations. The chip will also need to be maintained at an optimal
temperature of 37C for culturing. Finally a waste reservoir will be connected to the output port to collect the waste over the experimental procedure.
Characterization will follow a similar procedure to that of the YF1/FixJ plasmid with optical and data analysis. Additionally, pigmentation density will
be studied through characterization of different media, sugars, and temperature conditions.
YF1/FixJ Two Component Systems
pDawn Plasmid Containing the Engineered YF1/FixJ
Virtual Ladder Created in Benchling
Millifluidic Chemostat Design
Millifluidic Chemostat Chambers and Media Pass
2mm Polycarbonate Air Channels
2mm Polycarbonate Liquid Channels
70um pdms Control Layer
Yf1/FixJ and amilCP
mUAV Containing amilCP Gene
Loading Media
Millifluidic Chemostat Experimental Setup
Flushing Out
Prototype Testing
Prototype Fluid Flow