1.HBUT&BNU

In our collaboration with Beijing Normal University (BNU), we helped induce the expression of mCheery by salicylic acid(SA), measured its fluorescence intensity to determine the optimal concentration induced by SA and the optimal induction time at the optimal concentration. And the BNU-China helped us develop a mathematical model of the Nickel Hunter 2.0.

Their corresponding page can be accessed here: https://2018.igem.org/Team:BNU-China/Collaborations

HBUT: Experiment

Overview

Bioengineering uses stable, highly productive mutants, target strains, which contain foreign genes. However, screening these mutants costs vast time and workforce, and it is difficult to avoid using antibiotics. The philosophy of BNU-China team is to use salicylic acid to induce glucose dehydrogenase expression to enhance the strain's pentose phosphate pathway, giving the strain a growth advantage. EmrR protein can inhibit the role of the downstream promoter P_emrR, so that the gene downstream of P_emrR can not be expressed, but salicylic acid (SA) can bind to emrR to eliminate this inhibition. BNU-China team sets the gene of P_emrR downstream to gdh, using SA to control this route. After the BNU-China team replaced gdh with mCherry, we helped induce the expression of mCheery by SA, measured its fluorescence intensity to determine the optimal concentration induced by SA and the optimal induction time at the optimal concentration.

Protocol

I. Determination of the optimum concentration of SA for induction

Process:

1. Pick a single colony from the top of the board, expand it with liquid LB containing Amp, and control the time at about 11h.

2. Prepare for SA induction verification:

(1) The whole experiment can be done in 3-5 parallel groups. In the ultra-clean platform, the expanded liquid is divided into several 20ml large centrifuge tubes, each tube is filled with 2ml. Pay attention to the cover of the large centrifuge tube when filling the bacteria solution, wrap it with the sealing film, and tighten the rubber band (because the sealing film can be ventilated, if the cover is covered with oxygen, it will be insufficient).

(2) Adding SA, so that the concentration of SA in the system is 0 mg / L (ie, without SA), 0.001 mg / L, 0.01 mg / L, 0.1 mg / L, 0.3 mg / L, 0.5 mg/L, 1 mg/L.

(3) 37-degree shake flask culture induction for 5 h.

(4) All the samples and the blank E.coli K12 of the expanded culture were placed in the upper 96-well plate (operated in a clean bench), using ELIASA to measure the fluorescence intensity and OD of each sample well, and the fluorescence was mcherry red fluorescence. (Absorption wavelength 580 nm, emission wavelength 610 nm).

Results:

As shown in the picture, when the SA concentration is 0.3-0.5 mg/L, the maximum fluorescence intensity per unit OD can be detected, so the optimum concentration for SA induction is 0.3-0.5 mg/L.

II. Find the optimal induction time at the optimum concentration

Process

Find out the relationship between the time of induced expression and the fluorescence intensity at the SA concentration of 0.3 mg/L and 0.5 mg/L.

1. Expand the culture of puc19+emrr strain (note the addition of AMP resistance) for about 11 hours.

2. Set 3-5 parallel groups, in the ultra-clean platform, divided the expanded bacterial liquid into several 20ml large centrifuge tubes, each tube is filled with 2ml. Pay attention to the cover of the large centrifuge tube when filling the bacteria solution, wrap it with the sealing film, and tighten the rubber band(similar to the above).

3. Add SA to the system so that the SA concentration in all systems becomes the optimum.

4. Immediately measure the fluorescence intensity and OD value on a 96-well plate using ELIASA after the addition of SA.

5. Measure the fluorescence intensity and OD every 30 min, after 5h, record the experimental data.

6. Making the fluorescence intensity- time image of the unit OD and find the optimal induction time.

Result:

As shown in the picture, the fluorescence intensity per unit OD reached the highest at 3.5 h, so the optimum induction time at the optimum concentration is 3.5 h.

BNU：Modeling

Overview

Ni²⁺ is one of the heavy metal pollutants in our environment. In order to detect the concentration of the Ni²⁺, our team are going to use the NcrB protein operon to achieve this goal. In this system, the nikABCDE gene will express the NikABCDE protein, which belongs to the ATP-binding cassette transporter. It can transport Ni²⁺ from the environment into the cell consuming ATP at the same time. And Ni²⁺ is the signal we need to detect. At the same time the ncrB gene will express NcrB protein, which is a repressor combining with the pncrA promoter, inhibiting the expression of the downstream gene luxCDABE. However Ni²⁺ will bind with the NcrB protein, causing the NcrB protein drop from the pncrA promoter. In this way, the luxCDABE gene will express. And the LuxCDABE protein can give out light by consuming energy in the cell. Thus we can detect the concentration of Ni²⁺ by measuring the luminescence intensity.

Hypothesis

1. The hypothesis on the diffusion equilibrium of Ni²⁺

We assume that the ions distribute uniformly inside the cell immediately after they are transported into it through NikABCDE. As long as the speed of transportation is lower than the speed of diffusion, the ions are unlikely to aggregate around the transporter protein(e.g., NikABCDE). According to our knowledge, most of the transporter proteins do not possess such a high transporting efficiency, which means this hypothesis is valid in a larger degree.

2. The hypothesis on the relationship between NcrB concentration and the copy number of the recombinant vector

Since each of the recombinant vector contains only one copy of the target gene, we can assume that the vectors have the same ability of expressing NcrB. Furthermore, it’s reasonable to assume that the final concentration of NcrB is proportional to the copy number of the recombinant vector.

3. The hypothesis on the concentration of NcrB during the process in which Ni and pncrA bind with NcrB competitively

There are three subtypes of NcrB in the cell: the free state NcrB, the NcrB binding with pncrA and the NcrB binding with Ni²⁺. We assume that the concentration of total NcrB is not influenced by the expression of NcrB gene or its degradation process, because the speed of these two processes is relatively low compared with the competitively binding process. More importantly, based on this hypothesis, we can further assume that the concentration of free state NcrB remains constant during the process of competitive binding. This is because the kinetics feature of the process is similar to enzymatic reactions.

Notations

Ki: the reaction rate constant of each reaction

Ni: the concentration of Ni²⁺ inside the cell

Ne: the concentration of Ni²⁺ outside the cell

E: the concentration of the Ni²⁺ transporter protein NikABCDE

P: the promoter pncrA which binds with NcrB

B: the copy number of the recombinant plasmid in a single cell

LI: luminescence intensity

The construction and the solution of the math model

According to the hypotheses proposed above, the following dynamic

process exists inside the engineering bacteria:

1. The diffusion of Ni²⁺

We described this process through the differential equations below:

This system of equations is a set of first-order linear constant coefficient differential equations, and this system of equations has analytical solutions.

2. The transcription of LuxCDABE conducted by NcrB

Since the concentration of the free state NcrB remains constant, and the concentration of total NcrB is a function of the copy number of the plasmid, we can present the following equations:

Furthermore, we know that there is an equilibrium in the process of NcrB binding with Ni, which can be stated as follows:

As is proposed above, LI is linearly dependent on the number of free state promoter. That is to say,

Substitute 2.4 into 2.5, we can get the following equation:

Further substitute 2.6 into 1.4, now we have obtained the final expression of LI, in which time and the concentration of Ni2+ are the variables.

Discussion:

1. Luminescence intensity versus time curve with a constant concentration of Ni2+ ions

According to the relevant data in the literature and obtained by experimental measurements, we roughly made a function image of the brightness signal as a function of time, and the image is as follows.

2. Relationship between final luminescence intensity and Ni2+ ion

2. CCiC

From 28th Aug to 31th Aug, the 5th CCiC(Conference of China iGEMer Community) was host in Shanghai. Our team was honored to be invited. More than 50 iGEM teams in China attend the meeting, and we had heated discussion about synthetic biology with each other. During the meeting, we demonstrated our project, from the design to the preliminary results. iGEMers from other teams put forward some constructive suggestions, and we also gained some collaborations opportunities from them. We were happy to make friends with some iGEMers and we stayed in contact with each other even after the meeting, which contributes to integrated collaboration. CCiC is really a big event for iGEMers in China. see more

3. OUC-China

In this year’s collaboration, the OUC-China team offered us a comic book about synthetic biology. The book exhibited the complex biology model and principle with funny comics, making it more easier for students to read and understand. So when we went to the Wuhan NO.20 high school classes to communicate with them, we brought the comic book with us, and hoped to bring the young students together to appreciate the fun of biological knowledge. Firstly we introduced in the form of comics, and brought them into the world of biology. We also used this comic book for attracting and recruiting new members for our next year’s competition.

During our voluntary project and introductory courses this comic book greatly assisted us to introduce synthetic biology to the public. In turn, we helped to make a detailed investigation and collect the reader’s opinions about this book, we really hope to make this comic book better to help more people appreciate the charm of synthetic biology.

4. HUBU-Wuhan

We established a close relationship with HUBU-Wuhan. It is the first time for them to participate in the iGEM competition. We have two formal meetings with HUBU, the first one on December 1, 2017, and the second one on July 13th, 2018. Besides, there are continuous communication along with the whole process of our iGEM project. In the second meeting, we went to their the conference room, listening to their weekly report. In addition, our PI, Jun Dai also gave valuable suggestions about how to improve their project. Our latest meeting was on 31th Aug, during the 5th CCiC in Shanghaitech University, we shared our latest process of the project, also appreciated each other’s posters as well.

Their corresponding page can be accessed here:https://2018.igem.org/Team:HUBU-Wuhan/Collaborations