Team:NJAU China/Design

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The Donor Cell and The “Write-in” Plasmid

Blue Light Sensor

1. Background:
The unique physical properties of the environmental factor light allow for an independent photocontrol of various microbial processes. The blue light sensor based on LOV (light, oxygen, or voltage) domain has been designed1. The blue light-sensing protein YF1 is like a switch, which can be unphosphorylated by blue light and back to the phosphorylated state in black. When phosphorylated, YF1 passes a phosphoryl group to FixJ, which then binds to and activates FixK2 promoter 1. We use a logical NOT gate cI repressor to invert the response of the (NOT blue) sensor to make sure that of a blue light sensor. The chimeric light sensor YF1 was created by fusing the light perception domain of YtvA from the B. subtilis to the FixL kinase domain from the Bradyrhizobium japonicum1. YtvA acts as a blue-light photoreceptor in the environmental branch of the general stress response pathway and carries a blue-light-sensitive flavin-binding LOV domain2, 3. The response regulator FixJ forms a two-component system together with FixL4. FixK2 promoter is the wild-type promoter to which phospho-FixJ binds. FixJ in turn can be regulated by YF1, the blue light-sensing protein.
2. Introduction:

Blue light sensor is in the write-in system. When the blue light signal is on, it will express TraI to transfer the plasmid. So, the information is stored. With a constitutive promoter, the blue light-sensing protein YF1 can

persistently sense the light. As the transcription from the sensor is repressed by blue light and activated in dark, we use a not logical gate, repressor cI to ensure the system is activated by blue light.

3. Reference:
[1] Moglich, A., Ayers, R. A. & Moffat, K. (2009). Design and signaling mechanism of light-regulated histidine kinases. J. Mol. Biol. 385, 1433–1444.
[2] Losi A (2004) the bacterial counterparts of plant phototropins. Photochem Photobiol Sci 3(6):566–574
[3] Losi A, Polverini E, Quest B, Gärtner W (2002) First evidence for phototropin-related blue-light receptors in prokaryotes. Biophys J 82(5):2627–2634
[4] Fischer HM (1994) Genetic regulation of nitrogen fixation in Rhizobia. Microbiol Rev 58(3):352–386

TraI——An Essential Helicasein Transfer

1. Introduction:
The TraI protein is known as an ATP-dependent helicase which requiresa single-stranded region of DNA of about 200 nt as a substrate1.More recently, theTraI protein has been demonstrated to be required for nickingat oriT2.In our system, we try to control its expression to control the process of conjugation, which represent the process of write-in.
2. Reference:
[1] Abdel-Monem M, Taucher-Scholz G, Klinkert M Q. Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor[J]. Proceedings of the National Academy of Sciences, 1983, 80(15): 4659-4663.
[2] Traxler B A, Minkley E G. Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein [J]. Journal of molecular biology, 1988, 204(1): 205-209.

The Shuttle Plasmid

1. OriT (Origin of Transfer):
Unlike other plasmids, the conjugative plasmid has its own origin of transfer nic region. This is a specialized portion of the genome where one of the two strands of the circular plasmid DNA is cut to allow for rolling circle replication of the plasmid containing the oriT region into a recipient cell. During rolling circle replication, the cut end of one strand is inserted into the recipient cell while recipient polymerases construct the complementary strand back into a circular piece of DNA. Schematic diagram of bacterial conjugation. 1 - Donor cell produces pilus. 2 - Pilus attaches to recipient cell, brings the two cells together. 3 - The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell. 4 - Both cells circularize their plasmids, synthesize second strands, and reproduce pili; both cells are now viable donors. Image courtesy of Wikipedia.
2. T7 promoter +RBS+GFP+terminator: part: BBa_I746907
It is driven by a T7 promoter. It will express Green Fluorescent Protein (GFP) while in E.coli BL21.
3.OriT+ T7 promoter +RBS+GFP+terminator (the shuttle plasmid)
This part is called transfer plasmid. It will be transferred from donor cell to recipient cell by bacterial conjugation. And it will express Green Fluorescent Protein (GFP) while in E.coli BL21.

The Recipient Cell and the “Erase” Plasmid

Red light sensor

1. Background:
Light is an efficient and convenient way to control gene expression in microbes without chemical toxic or separating troubles. The engineered red-light-regulated gene expression system has been reported1. The red light-sensing protein Cph8 is like a switch, which can be dephosphorylated by 650-nm light and back to the phosphorylated state by 705-nm light. When phosphorylated, Cph8 passes a phosphoryl group to OmpR, which then binds to and activates transcription from PompC2. Because it is inactivated by red light, Cph8 can be considered a logical (NOT red) sensor. A genetic inverter or logical NOT gate is used to invert the response of the (NOT red) sensor to that of a red light sensor3. Here, we choose repressor TetR. The chimaera Cph8 is the phytochrome Cph1 from Synechocystis PCC6803 fusing with a cyanobacterial photoreceptor EnvZ which can engineer a red light-regulated transcription system in E. coli, with the help of Phycocyanobilin (PCB). EnvZ–OmpR is a well-studied two-component system, which normally regulates porin expression in response to osmotic shock2. Phycocyanobilin, the part of the photoreceptor that responds to light is biosynthesized by genes (ho1 and pcyA) from Synechocystis that convert haem into phycocyanobilin (PCB)4.
2. Introduction:
Red light sensor is in the erase system. When the red light signal is on, it will start CRISPR up to cut the transferred plasmid. As a result, the information stored is deleted. With a constitutive promoter, the red light-sensing protein Cph8 can persistently sense the light. As the transcription from the sensor is repressed by red light and activated by dark, we use a not logical gate, repressor TetR to ensure the system is activated by red light.
3. Reference:
[1] Levskaya, A., Chevalier, A. A., Tabor, J. J., Simpson, Z. B., Lavery, L. A., Levy, M. et al. (2005). Nature, 438, 441–442.
[2] Utsumi, R. et al. (1989). Science 245, 1246–1249.
[3] Tabor, J. J. et al., (2010).Multichromatic Control of Gene Expression in Escherichia coli. J. Mol. Biol.
[4] Gambetta, G. A. & Lagarias, J. C. Proc. Natl Acad. (2001). Sci. USA 98, 10566–10571.

The CRISPR System Aimed for Erasing

1. Background:
In procaryotes, CRISPR-Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. Nowadays scientists have reformed this system as an RNA-guided nuclease for genome editing especially typeⅡ1. TypeⅡCRISPER-Cas system is consist of cas9 protein and a special sgRNA. Through the sgRNA, cas9 protein can thus be re-directed toward the target sequences and cut them.
2. Introduction:
In our design, CRISPER-Cas9 system is the main part of Erase-plasmid. Cas9 protein is under control of one weak constitutive promoter. With the right light induced, GFP sgRNA sequence will be transferred. Then Cas9 protein will bind to GFP and cut it, thus the Shuttle-plasmid will be degraded which means “erase”.
3. Reference:
[1] Ran, F. A., et al. 2013.Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281-308.

The Whole System

Our system simulates the storage and erasure of computer binary information completely. When using blue light irradiation (Represent the appearance of the signal),the TraI Protein will be expressed in the donor cell, and the conjugation process begins.After the shuttle plasmid was reached in the recipient cell, and under the action of T7 RNA polymerase, a large number of green fluorescent protein will be expressed in the recipient cell. So far, the bacterial system has been changed from the previous non fluorescent state to a fluorescent state. We can identify the system to complete the memory of an instantaneous information.When using red light irradiation (Represents the erase instruction), the CRISPR system will start to work. It will shear the gene of GFP on the shuttle plasmid using Cas9. Because the linear DNA in the bacteria will soon be degraded, and with the loss of gene and the degradation of the original protein,the bacterial system will change from the fluorescent state to a non-fluorescent state. We can identify the bacterial system to complete the erasure of a piece of information. At this point, the whole system has experienced a cycle process.And if the system is to implement the "blue - red" repeated irradiation, the whole process will be repeated again and again. Here is a Sketch Map which shows the whole system about our project.

A New Measuring Device

The hardware part of our project, this device, is used to control the writing and erasing process (by emitting blue or red light) and monitor the state of the system (by detecting the fluorescence level).
-The schematic:

-The photodiode and I/v converter:
Photodiode is a semiconductor device that converts light into current. The light current is extreme low (<1E-6A, Max).So, the op-amp must have ultra-low input bias current (< 1pA).
-The I/v converter:

Vout = I * R -ADC:
Analog-to-digital converter converts voltage to a digital number. Number can be received by Arduino and sent to computer for further analysis.
The following part shows the Arduino sketch which obtains data and controls LEDs. Notice that we set to get readings with and without UV LED turning on. And the final result
code download
Here shows the processing sketch running on the pc. Function of this program is to collecting data and forming a csv file to save them. Also, it sets all the variables, like number of scans for each measuring, skipping milliseconds for every measuring, repeat and interval between measuring.
It shows the last measuring’s every scan readings, from that we can get a brief knowledge about the noise.
code download
Some circuit (incomplete)