How to obtain our target metallo-beta-lactamases
Genetic Circuit Design
The very first step of our project is to attain the protein, that is to say, to build gene expression
circuits, consisting of promoter, tag, target sequence, and other necessary parts.
After conducting a series of permutation and combination, we used our expression system engineering bacteria E.coli to assess fitness of circuits on whether the circuit can express the protein smoothly, whether the protein form an inclusion body, whether the protein has considerable enzyme activity and so on. In this way we can find the most adaptive circuit.
After we tested circuits made up of only T7 promotor, His tag and beta-lactamase, we disappointedly
the effect unsatisfactory. The expression quantity is too low to take further purification or the
protein all become inclusion body. Through our analysis, we discover we probably need a tag which
improves dissolution or stimulates expression quantity.
The way to test circuits is to transform them into E.coli engineering bacteria to find out if it can work appropriately. A successful circuit can perform and provide soluble target protein to us, however, most circuits and expression system mismatch so they are not able to produce useful protein then iterations exist. The “proteinality” matters.
How to find arrows targeting MBLs
Establishment of Fluorescent HTS system
Mechanism of Fluorescent Probe CDC-1
Consisting of three parts, CDC-1 is a extend-spectrum fluorescent probe which can be used for
the role of beta-lactamases. A lactam ring is designed as the hydrolyze site, a --CH2 plays the role
a bridge and an umbelliferone as the activatable fluorophore when hydrolyzing based on FRET.
According to reports, there are several detecting substrate that have been designed, specifically and extend-spectrally. Due to what we’ve chosen to express are subclass B1 and B3 beta-lactamases, CDC-1 is suitable to detect in HTS system.
The very first thing in the process of HTS system establishment is to determine suitable enzyme
concentration. When screening, a 96-well plate is used so that the reaction speed should not be fast
order to guarantee reaction holds on after mixing all wells. Also, in order to calculate the initial
velocity, the relationship of fluorescent intensity and time should be linear. Last but not least,
values of fluorescent intensity should not be very small to provide more accurate results.
So in order to explore the proper concentration of enzyme, we set 3 dilute gradients for large span. Then around the most fit one set more gradients for smaller span and plot the fluorescent intensity–time curve. All assays are repeated in triplicate at room temperature(around 25°C) and the baseline of blank controls is subtracted.
Buffer condition is also a key component in HTS system, owing to its huge effect on enzyme activity.
After collecting and reading articles about purification of beta-lactamases, we choose PBS as MBLs’
reaction circumstance. Also, we added Zn2+ into system as MBLs depends on it.
However, the concentration of each component should be optimized.
Therefore, this is a multivariate experiment. All the experimental test groups are designed following
the “N+N-1” rule, avoiding setting unnecessary groups.
Chart 1 Experimental groups of buffer conditions
*means standard value, the concentration of NDM-23 is relevant to EC80 value(see this part in Test), NaCl and pH value is relevant to PBS solution and Zn2+ is relevant to reports.
Index for determination of enzyme concentration: R2, EC80
In order to calculate the initial velocity, the relationship of fluorescent intensity and time should
linear fit. R2 (Coefficient of determination) is computed as a value
0 and 1. The higher the
value, the better the linear fit.
Values of fluorescent intensity should not be very small to provide more accurate results. When testing in different enzyme concentrations, calculate fluorescent rE (rate of Emission), plot rE with log[E] then get the value of EC80. Choose the concentration which is most close to EC80 value.
Index for determination of substrate concentration: Km, kcat
With CDC-1 as a substrate, we will measure the kinetic parameters of metallo-beta-lactamases by
Michaelis-Menten plots and Lineweaver-Burk plots and the Michaelis-constant (Km) and catalytic
constants (kcat) are determined. The catalytic efficiency (kcat/Km) of MBLs will be calculated.
data confirm the high efficiency of these enzymes in promoting the hydrolysis of CDC-1, which
guarantees a high detection sensitivity of CDC-1.
Additionally, when testing Km constants the concentration of substrate is 8.5μM due to storage necessity. And its value should less than or equal to Km value in HTS system to ensure a mixed order reaction.
Index for entire screening system: Z'-factor
In order to validate the stability and reliability of this HTS system, a factor called Z’-factor is
introduced to assess. It is a relative index to distinguish the target signal with background
The range of Z’-factor can be less than zero to 1. If it is more than 0.5, it indicates that this
model can be applied in high-throughput screening. 
Equation 1. How to calculate Z’-factor
How to evaluate the role of arrows
Methods to evaluate therapeutic effects
We will examine whether the antibiotic is hydrolyzed by beta-lactamase by UV-vis spectroscopy. According to the degree of decomposition of cefazolin, the inhibition rate of the corresponding inhibitor was calculated, and the IC50 of the inhibitor combined with cefazolin in the condition of live bacteria was obtained through the inhibition rate, so as to evaluate the therapeutic effect of dual drug combination.
Beta-lactam antibiotics inhibit bacterial cell wall formation by binding to the bacterial transpeptidase via the c-n bond on the beta-lactam ring. beta-lactamase hydrolyzes the C-N bond of the beta-lactam ring causing antibiotics to fail. The result of hydrolyzing cephalosporins is a decrease of the absorption characteristic of the C–N bond (typically at ~260 nm), which can be observed by UV-Vis spectroscopy. We take advantage of this spectroscopic change to determine if the antibiotic is broken down. 
Determination of antibiotic concentration and bacterial solution concentration in the reaction system
We chose 250 μM of cefazolin, because the peak concentration of cefazolin in human serum is 250 μM, and using this value can make our evaluation of therapeutic effect closer to human condition.  the same time, we conducted orthogonal experiments on the induction time and concentration of bacterial strain, used EDTA as a positive control to test the decomposition capacity of bacterial strain, measured the IC50 value of EDTA, and compared it with the literature value of the drug-resistant strain producing beta-lactamase naturally, and selected the determination condition closer to the literature value as the standard for the preparation of bacterial solution.
Negative and Blank Controls
To ensure the accuracy, we designed the following experiment as controls.
1. The extracted beta-lactamase protein was mixed with antibiotics, and the absorbance value of the specific absorption peak obtained by the antibiotics was continuously measured to see whether there was a trend of decrease.
2. E. coli with the MBL gene was mixed with antibiotics and specific absorption peaks of antibiotics were determined to demonstrate the expression and function of beta-lactamase in E. coli.
3. E. coli without the beta-lactamase gene and the buffer used to prepare Escherichia coli suspension was mixed with the antibiotics separately and specific absorption peaks of the antibiotics were continuously determined to prove that beta-lactamase broke down the antibiotics.
4. We mixed several lactam and non-lactam kinds of antibiotics with E. coli strains that had transferred with beta-lactamase separately, and determined the specific absorption peaks of each antibiotic to prove that our enzyme was indeed beta-lactamase.
Index for assessment of inhibitory ability: IC50
Different concentrations of inhibitors were added into system to plot the curve of inhibitory rate to log[I]. In vitro, substrate CDC-1 was still used in system to demonstrate the change of fluorescent intensity. However, the initial value and final value of absorbance displayed by antibiotics were calculated to show the change of absorbance value using Nanodrop microspectrophotometer in vivo. 
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