Team:NCKU Tainan/Contribution


Contribution

Aid development of related research

Overview

During the development process of Eye kNOw and Eye Screen, we realized we need to find ways to prove our success. Thus, we have done some experiments and collected the data, including the activation of heat-inactivated tetracycline and chlortetracycline, assembling function generator, DIC, and 3D printing. We organized our research data and information to speed up the development of related research projects. Not only that, we hope that our contributions can aid other iGEM teams and use it to improve their projects further. Through these efforts, we believe that we are able to make a significant contribution to boost and inspire more people to help and make our world a better place.

Tetracycline, chlortetracycline

Our first kill switch design used TetR fused with VP16, tet-off system[1] to control the Colicin E7(BBa_K117000) as our biosafety measure. We will put tetracycline analog into the contact lens, this will prevent the VP16 to activate the lysis gene. If the bacteria leave the contact lens, where the environment has low concentration of tetracycline analog, the lysis gene will be activated.(Fig. 1)

There are several options when it comes to tetracycline analog that can act on the tetR protein, changing its DNA binding affinity, however, according to Biomol GmbH-Life Science Shop, 50 mg of Anhydrotetracycline costs 54 US dollars.[2] In search of a cheaper alternative, we turned to tetracycline (TC)[3] and chlortetracycline (CTC)[4], which has been reported to bind to tetR protein and affect its affinity to tetO.

Fig. 1. Design schematic of the killswitch using tet-off system.

After many attempts constructing this plasmid resulted in failure, we turned to gene knockout as our biosafety. However, we can still characterize the effect of TC and CTC on TetR using iGEM BioBrick BBa-K611059.(Fig. 2)

Fig. 2. iGEM BioBrick BBa-K611059.

BBa-K611059 has two open reading frames: a pTet promoter controlled GFP, and pBAD promoter controlled TetR. As we use arabinose to produce TetR to repress pTet, this allowed us to try different tetracycline analogs and observe GFP signal recovery to assess their potency. Since most tetracycline analogs have antimicrobial activity, we test two different ways to compensate for this effect, heat inactivation of tetracycline analogs and co-transform tetracycline resistance plasmid.

We first transformed the plasmid carrying bioBrick BBa-K611059 into BW25113, which cannot metabolize arabinose, and added different concentrations of heat inactivated CTC[5] to see if the GFP signal is restored.

After 12hr of culture with different concentrations of heat-inactivated CTC and 0.0001% of arabinose, we can see as arabinose was added into the culture, the normalized GFP signal slightly decreased, suggesting BBa-K611059 functions correctly. As 20µg/ml heat-inactivated CTC can rescue the GFP signal, and 30µg/ml will result in higher GFP signal. Also, through the OD600 results we can also conclude the heat-inactivated CTC does not affect E. coli growth.(Fig. 3)

Fig. 3. GFP value according to different concentrations of heat inactivated CTC with 0.0001% of arabinose and the OD600 value.

Despite showing that heat-inactivated CTC can activate pTet, we were not satisfied with the dynamic range, so we decided to challenge using TC itself, since this operon originated in response to antibiotic resistance. In concerns of TC will be deadly to E. coli itself, also there has not been reported heat-inactivation TC can activate pTet, we co-transformed a TC resistance plasmid, pACYC184, into BW25113 to conduct further experiment.(Fig. 4)

Fig. 4. (A) GFP value according to different concentrations of TC with 0.002% of arabinose and the OD600 value; (B) GFP value according to different concentrations of TC with 0.0002% of arabinose and the OD600 value

We first tried two different arabinose concentrations, 0.002% and 0.0002%, both concentrations show repress GFP signal. However, bacteria experienced growth retardation in 0.002% of arabinose, we speculate that the high arabinose concentration induces osmotic stress to the bacteria. Also we can see as TC concentration increases, although the GFP signal remains the same, the OD600 decreases.This indicates that TC can induce pTet to produce GFP but suppress bacteria growth. We speculate that pACYC184 can not withstand such high concentration of TC.

From these results, since both TC and heat-inactivated CTC can activate pTet, but TC seems to affect cell growth at higher concentrations although we supplied the bacteria with resistance plasmid, we can conclude that heat-inactivated CTC is a better inducer compared to TC.

DIY function generator

How to make a function generator by yourself?

A function generator is a device used to generate different types of electrical waveforms over a wide range of frequencies. In our project, we need a function generator to trigger 40kHZ transducer.

However, for a portable device, a typical function generator is heavy and expensive. Therefore, we decided to make a function generator on our own!

We used the AD5930[6] to implement the function generator. It’s a chip that can generate waveforms with programmable frequency sweep and output burst capability by using Arduino Mega 2560 programming control, AD5930 can be operated in a variety of modes. The following is the production process and control methods.

Production process

  1. Draw the circuit diagram against the AD5930 datasheet. (The pins can be deleted according to functional requirements.)

    Fig. 5. Schematic diagram of function generator.

  2. Using Digital Experience Platform(DXP) to do layout.

    Fig. 6. DXP layout of function generator

  3. Print out the PCB board and solder.

    Fig. 7. Function generator (before and after soldering).

  4. Complete after testing!

    Fig. 8. Waveform generated by function generator (sinusoidal/triangle).

Control methods

Using Arduino Mega 2560 programming control, AD5930 can be operated in a variety of modes. To address the control register, D15 to D12 of the 16-bit serial word must be set to 0.

  1. Output waveform: Sinusoidal/Triangle waveform.

    Controlled by SINE/TRI (bit 9).

    • When SINE/TRI = 1, the SIN ROM is used to convert the phase information into amplitude information resulting in a sinusoidal signal at the output.

    • When SINE/TRI = 0, the SIN ROM is bypassed, resulting in a triangular (up-down) output from the DAC.

  2. Output Mode: Continuous/Burst mode.

    Controlled by CW/BURST (bit 7).

    • When CW/BURST = 1, the AD5930 outputs each frequency continuously for the length of time or number of output waveform cycles specified in the appropriate register, TBURST.

    • When CW/BURST = 0, the AD5930 bursts each frequency for the length of time/number of cycles specified in the burst register, TBURST.

  3. Output Frequency.

  4. Output cycle number (in Burst mode).

    there are two ways to control:

    • Based on clock period

    • Based on output waveform cycle

The complete code is as follows:

Digital image correlation (DIC)

Digital image correlation (DIC) is a method that analyzes the position of the spot on the surface of the analyte. We can obtain data from deformation, force, etc., according to the needs of the experiment.[7]

Software:

Vic-Snap 2010, Vic-3D 2010

Steps:

  1. Use powder to create spots. Since we have a black background, we use white spots to increase the clarity of the image. (The photo is presented in grayscale.)

  2. Take (A) calibration images, speckle images and import them into the software.

  3. Calibrate the software.

  4. (A) Select analysis location and (B) create the marker.

  5. (A)Adjust parameters based on (B) reference values.

  6. (A) Analyze the image and (B) data.

Fig. 9. User Interface of VIC-3D.

For other detailed operation methods, please refer to the website[8], it includes the reference manual and the solutions of common problems.

3D printing

3D printing, also known as additive manufacturing, created a solid three-dimensional object via the accumulation of different layers of materials laid down sequentially in different shapes[9]. Instead of the traditional method based on the removal of materials from a larger body, 3D printing allows for low consumption of materials, quick-turn functional prototypes, and a cost-effective process.

Applications of 3D printing are emerging almost by the day, as this technology continues to penetrate more widely and deeply across various fields, including industry, medicine, and architecture, and so on. For example, 3D printing's use in education, enabled students to realize their thoughts out of the computer screen and into the real world. Thus, our team came up with Eye Screen, a low-cost, non-invasive, and portable tonometer accessible to anyone.

This short introduction gives an overview of the things we had done and consider over the past few months of printing.

Software:

3D printing works by starting with a digital model in a 3D CAD (Computer Aided Design) file and then creating a physical three-dimensional object.

We design the complete device on the CAD software Solidworks that adopted in the process of 3D printing as it allows us to save our models in an STL format; the format needed for a 3D model to be processed by 3D printing software. It would be noted that the dimensional accuracy should be considered in the designing process.

Fig. 10. Illustration of Eye Screen holder.

Printing:

One of the key factors in 3D printing is the selection of material. PLA is a biodegradable plastic, derived from renewable resources like corn starch or sugar cane, which becomes the material we used.[10]

Fig. 11. Prototype of Eye Screen holder.

References

  1. Kim, H J, et al. “Tetracycline Repressor-Regulated Gene Repression in Recombinant Human Cytomegalovirus.” Journal of Virology. vol. 69, no. 4, 1995, pp. 2565–2573.
  2. Anhydrotetracycline (hydrochloride). Biomol GmbH - Life Science Shop. Published 2020. Accessed October 25, 2020. https://www.biomol.com/products/chemicals/antibiotics/anhydrotetracycline-hydrochloride-cay10009542-50
  3. Aleksandrov A, Schuldt L, Hinrichs W, Simonson T. Tetracycline-Tet Repressor Binding Specificity: Insights from Experiments and Simulations. Biophysical Journal. 2009;97(10):2829-2838. doi:10.1016/j.bpj.2009.08.050
  4. Bochner BR, Huang HC, Schieven GL, Ames BN. Positive selection for loss of tetracycline resistance. Journal of bacteriology. 1980;143(2):926-933. doi:10.1128/JB.143.2.926-933.1980
  5. Pósfai G, Koob MD, Kirkpatrick HA, Blattner FR. Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. Journal of bacteriology. 1997;179(13):4426-4428. doi:10.1128/jb.179.13.4426-4428.1997
  6. AD5930 Datasheet and Product Info | Analog Devices. Analog.com. https://www.analog.com/en/products/ad5930.html#product-overview. Published 2014. Accessed October 14, 2020.
  7. Digital image correlation and tracking, wiki; Retrieved from https://en.wikipedia.org/wiki/Digital_image_correlation_and_tracking
  8. Correlated SOLUTIONS; Retrieved from http://correlatedsolutions.com/support/index.php?
  9. Everything you need to know about Polylactic Acid (PLA); Retrieved from https://www.creativemechanisms.com/blog/learn-about-polylactic-acid-pla-prototypes
  10. What Is 3D Printing Technology – How It Works, Applications & Obstacles; Retrieved from https://www.moneycrashers.com/3d-printing-technology-applications-obstacles/

e-mail : igem.club@gs.ncku.edu.tw
phone : 06-2757575