MIT Class Site
Laura Maria Gonzalez
April 12, 2021
For my final project I am interested in creating light sensor that is responsive to UV light to create dynamic shading system for glazing in windows. The project
was inspired by the bacterial photography 2004 iGem proposal where sequences from a cyanobacteria and E.coli are combined to create a photoreceptive E.coli
that reveals a message in the presence of light. For a dynamic shading application the E.coli would respond to different levels of UV light revealing an opaque
color screen when triggered that would provide shading through a glazing microfluidic device. If the E.coli are able to also differentiate between different levels of
UV then different colors could also be deployed to alert people walking by of the intensity of UV as they pass by.

For this week I was curious if a similar light biosensor could be created through a cell-free system. To guide me through the assignment, I used the paper
"Bringing Light into Cell-Free Expression" (Zhang et al., 2020) The paper looked into developing a switch for dynamic control of transcription/translation process
through blue light. Advantages to a cell-free system include activating transcription/translation processes in vitro which can overcome biocontainment and
biosafety concerns.
What would your synthetic cell do? What is the input and what is the output.
Cell Free Optical Sensing. The synthetic cell would create a light (blue) sensing system by using light as an ideal control switch. Input: Blue Light
Output: Fluorescent Protein (mCherry)

Could this function be realized by cell free Tx/Tl alone, without encapsulation?
Encapsulation is still needed.

Could this function be realized by genetically modified natural cell?
Yes, in fact most optogenetic tools have been through genetically modified natural cells. This is because most optogenetic systems are usually based on
membrane-bound photo-reversible two component systems (TCSs) and require a synergistic effect of two proteins to achieve optical sensing. But cell free is
interesting application because of the biosafety and potential educational use / use as an optical cue in an artificial cell which might aid in creating a life-like
cell mimic.

Describe the desired outcome of your synthetic cell operation.
The desired outcome is to create a light control switch that reveals an image with blue light and hides the image in darkness.
What would the membrane be made of?
Phospholipids and cholesterol to make a lipid vesicle.

What would you encapsulate inside? Enzymes, small molecules.
Encapsulated inside is the cell-free reagents which include the tx/tl system, the E.coli extract and the pLight plasmid .

Which organism would your Tx/Tl system come from? Is Bacterial ok, or do you need mammalian system for some reason?
The Tx/Tl system would come from bacteria: E.coli.

How will your synthetic cell communicate with the environment?
The synthetic cell will communicate with the environment through Blue Light and a YF1/FixJ system. The system works through transcription regulation. The YF1/
FixJ system drives the expression of the phage repressor cI from the FixK2 promoter which represses the expression from the promotor pR enabling gene
expression in this case mCherry fluorescent protein under blue light.
List all lipids and genes
Lipids: POPC and cholesterol
Genes: YF1 and FixJ

How will you measure the function of your system?
Through visual cues from the fluorescent protein mCherry in regions that are exposed to light vs no fluorescent in areas left dark.
For part B of this week's assignment involved using cell-free systems to test different hypothesis about transcription and translation reactions. Transcription is
the process of transferring the information in DNA into a temporary copy called messenger RNA (mRNA). The name transcription makes sense because it means
the act of transcribing or making a copy. In order to successfully transcribe you need: (1) a promoter - indicates where transcription should start (2) a protein
coding sequence - the part of the nucleotide sequence that is transcribed to the mRNA and determines the order of the amino acids that will make up the
protein (3) a terminator - signals the end of transcription. Key to the process is RNA polymerase which binds to the promoter sequence and links together free
ribonucleotides to build a chain of RNA until it reaches the terminator sequence.

After transcription comes translation. Translation converts the information stored in mRNA into specific sequences of amino acids that are required for specific
proteins to be built. The mRNA nucleotides are read in groups of three (codons) resulting in a sequence of amino acids. Similar to transcription there is a start
and an end. The start codons signal the beginning of the protein coding sequence. A stop codon signals the end. Reading the codons is a ribosome which uses
transfer RNA (tRNA) to read the mRNA and link amino acids into a growing chain to build the protein. The name translation makes sense since the codons are
being translated into amino acids.

To see transcription and translation in action we made use of cell-free systems! This is advantageous because we can visualize transcription through
fluorescence via an aptamer. Aptamers bind with high affinity to small molecules to induce their fluorescence. For our experiment we will be using Broccoli RNA
which will fold into a specific structure that will bind to the ligand DFHBI which will fluoresce once bound to the folded Broccoli.
The experiment is based on the Biobits Kit. For the positive control I specified using DNA A which includes the aptamer and the red fluorescent protein allowing
us to visualize both green and red fluorescence. The negative control used water with no DNA since it does not include the parts necessary to fluoresce either
green or red therefore no color should appear.

For the transcription/translation tests I wanted to test three things:

(1) Rifamycin - an antibiotic that inhibits bacterial DNA-dependent RNA synthesis. It has high affinity for prokaryotic RNA polymerase. I also read that if rifamycin
binds to the polymerase after the chain extension, no inhibition is observed. Therefore I wanted to test initially including Rifamycin (1x), then introducing
Rifamycin after transcription occurs, and testing the high affinity through a low concentration (0.1x).

(2) Puromycin - an antibiotic that inhibits protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation.
The tests conducted with Puromycin focused on the effects of different concentration.

(3) UV Light - causes damage to DNA strands and should therefore affect the transcription/translation process.

The results confirmed some initial hypothesis. For example, the water caused no green or red florescent since neither transcription or translation occurred. The
positive control "standard" also worked demonstrating both green and red fluorescence. The UV light also effected the transcription/translation process. Visible
is a faint murky fluorescent which appears slightly orange in the image. Perhaps some transcription and translation occurred, but the damage to the DNA does
not allow it to fluoresce clearly and brightly.

A few unexpected results included the Puromycin which inhibited translation at both 0,1x and 10x concentration. I initially thought 0.1x would allow for partial
translation. The rifamycin also reminded me that timing inhibition at the molecular level is incredibly difficult. RNA transcription is over within milliseconds and
the cycles are continuously begin over again billions of times therefore you cannot target just translation even after we get a green fluorescence. This is
confirmed in the test results. Rifamycin introduced at different stages still produced the same results - no visible green or red fluorescence since RNA
synthesis was disrupted. For further tests I would be curious to learn more about the effect of UV light. For this round, I only tested 30 minutes but what would be
the effects at different time intervals such as 5 min or an hour.
Transcription/ Translation
Transcription/Translation Experiment Table
Electromagnetic Spectrum, Source
Blue Light Sensing Schematic, Zhang et al.
Transcription/Translation Experiment Results
Wavelength On = 430nm
Wavelength Off = Dark
Zhang, Peng, et al. "Bringing light into cell-free expression." ACS Synthetic Biology 9.8 (2020): 2144-2153.

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.

Khalil, Ahmad S., and James J. Collins. "Synthetic biology: applications come of age." Nature Reviews Genetics 11.5 (2010): 367-379.

Ramakrishnan, Prabha, and Jeffrey J. Tabor. "Repurposing synechocystis PCC6803 UirS–UirR as a UV-violet/green photoreversible transcriptional regulatory tool in E. coli." ACS synthetic biology 5.7 (2016): 733-740.