Team:NTHU Taiwan/Collaborations

Collaborations

Collaboration with NCTU

Overview

1. The suicide protein, ydfD, from NCTU_Formosa was characterized by O.D measurement, and we found its limitation of fully killing the bacteria.
2. We turned to physically way to address biosafety, by membrane filtration.
3. NCTU help us test the filter efficiency in return.

Introduction

NCTU_Formosa has always been the best partner through our project. Over the past four years, they had possessed projects related to agriculture problems. We had a closed relationship and frequently communicated with them on agriculture issues and applications. We designed meetups, and we invited them to hold a public speech with us. They introduced us to their advisor, who is now running an intelligence agriculture company, which influenced the way we design our sensing system.

Motivation

In 2019 collaboration, NTHU_Taiwan and NCTU_Formosa dedicated to the biosafety concern. As our Fertilizer Tank displays, we have to ensure neither can tunable-temperature-sensitive (TTS) bacteria escape nor can they survive when releasing to the environment. For NTHU Wet Lab, the first mindset of dealing with this challenge is using a typical suicide protein, ydfD. Therefore, the escaping bacteria from the two-chamber will die immediately. Fortunately, ydfD is a crucial part in NCTU iGEM project E. Phoenix, so we make an exchange for ydfD, test the suicide efficiencies by O.D value measurement, and complement the result of NCTU Wet Lab with these data.

However, it seems that the biological way is not effective enough to meet the biosafety concern. To guarantee no bacteria will break out, NTHU_Taiwan turns to a physical method, filtration by a membrane with a smaller pore size (7~5 μm ). Theoretically, if the silver-immobilized membrane is available, TTS bacteria can be filtered out and killed at the same time.

For NCTU

From NCTU

Protocol of ydfD function test

Purposes

To test the toxicity of each of our toxin gene by observing the growth curve of E. coli BL21 (DE3) after induction.

Strain

E. coli BL21 (DE3) with pSB1A3 containing insert part Plac-RBS-ydfD-Term.

Methods

1. Pick one colony (in common size) and incubate it for 12~16hr.
2. Dilute the culture 50 times and divide them into 6mL broth. Keep detecting the O.D. value every 30 minutes.
3. Add 500μM of IPTG when O.D. value reaches 0.3.
4. Load the sample into 96-well plate and set the plate reader for long-time detection, the reader incubates at 37°C and detect the O.D. value every 5 minutes.

Experiments

Figure 1. Exchange ydfD from NCTU_Formosa lab

Figure 2. Running OD600 measurement for NCTU_Formosa

Results

Raw Data

Figure 3. Growth curve with suicide protein

Protocols of Membrane Test

Introduction

We will investigate the filtering effect under O.D 0.7 of E.coli.

Figure 1. NCTU_Formosa leader, Ryan, holds the membrane after basic treatment

Figure 2. Hydrophilic membrane in fertilizer tank

Materials

• We will investigate the filtering effect under O.D 0.7 of E.coli.
• Hydrophilic membrane within a diameter of 5 cm
• Fertilizer Tank designed by NTHU_Taiwan

Procedures

1. Prepare the filter and R0010 E.coli ( O.D 0.7)
2. Fill the FA chamber with culture and with a total height of 10 cm
3. Collect the filtrate and PCR check every 10 minutes, and the PCR formula is 2 µL filtrate + 3 µL primer mixture (VF2, VR, water) + 5 µL taq
4. Document the filtrate volume and the PCR result

Results

While NCTU Formosa tested our membrane in their lab, they found out that it is too vulnerable that they can barely use it. Moreover, when they immerse the membrane into basic solutions--which the membrane will be water-soluble and can prevent bacteria, as well as fatty acid, leaking out the Fertilizer Tank--the membrane embrittles and is unable to filter. They reported their problem to us, and together we did the same experiment in our lab again. We discovered that the problem on the vulnerability of the membrane seems unsolvable, which makes it hard to apply in our Fertilizer Tank. Hence, NTHU Taiwan decided to use another kind of hydrophilic membrane as our filter to meet the concern of biosafety.

Collaboration with USTC

In CCiC, we had communicated with lots of teams. One of the teams we went beyond communication, and constructed collaboration is the USTC team.

Introduction

Azo dyes have been widely utilized in the coloring of various kinds of material, such as fiber, leather, and paper. However, due to its high stability in the aquatic environment, azo dyes are difficult to deal with. It has imposed a long-term adverse effect on the environment. Besides the toxicity to the aquatic creatures, azo dyes will also cause water pollution and it is detrimental to human health.

Meanwhile, there isn’t any existing microbial or chemicals treatment that can tackle the azo dyes efficiently. Thus, the innovative USTC team is determined to solve this unmet environmental problem. They introduced a degradation system into the bacteria, and this system is cloned from a species of bacteria that is capable of generating electron. The reducing power of this system engineered within the azo-dyes resistant bacteria is a great combination to deal with the azo dyes.

Motivation

When they introduce the electron transfer module into the bacteria, a QS regulator was coupled to repress the expression of the four genes related to electron transfer initially. But the widely used Lux promoters have a high leakage that the baseline expression will affect the degradation efficiency. Thus, they had to improve the Plux promoter.

Result

Hence, the USTC team communicated with us. We mentored them and acknowledge them the basic promoter improvement method and how to carry them out. We enhanced their design.

References

1. Virus disinfection in water by biogenic silver immobilized in polyvinylidene fluoride membranes Water Res. 2011 Feb;45(4):1856-64. doi: 10.1016/j.watres.2010.11.046. Epub 2010 Dec 7.