Team:TJUSLS China/Design
Design
Overview
Issues
Since antibiotic-resistant bacteria producing beta-lactamases spread widely around the world, it is urgently needed to find novel therapies against diseases related to beta-lactamases. Compared with design and synthesis from beginning of new compounds which can be used in beta-lactam/beta-lactamase inhibitors antibiotic-resistant drugs, to find naturally existing inhibitors is a faster way.
Solutions
Some new-type fluorescent probes have been reported in assays detecting beta-lactamases. Now that it can reflect the activity of beta-lactamases, fluorescent intensity would decrease as soon as extent inhibitors compounds play roles. Thus it performs a direct way to high-throughput screen potential inhibitors of metallo-beta-lactamases. Based on synthetic biology methods, we design our parts to realize our project goals, including inhibitors screening and assessment.
How to obtain our target metallo-beta-lactamases
Genetic Circuit Design
Principles
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.
Iterations
After we tested circuits made up of only T7 promotor, His tag and beta-lactamase, we disappointedly
found
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.
Test
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
Principles
Mechanism of Fluorescent Probe CDC-1[1]
Consisting of three parts, CDC-1 is a extend-spectrum fluorescent probe which can be used for
reflecting
the role of beta-lactamases. A lactam ring is designed as the hydrolyze site, a --CH2 plays the role
as
a bridge and an umbelliferone as the activatable fluorophore when hydrolyzing based on FRET.
According to reports[2], 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.
Pre-experiment
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
in
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.
N+N-1 Principle
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.[1]
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.
Test
Index for determination of enzyme concentration: R2, EC80
In order to calculate the initial velocity, the relationship of fluorescent intensity and time should
be
linear fit. R2 (Coefficient of determination) is computed as a value
between
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.
These
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[4]
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
groups.
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. [5]
Equation 1. How to calculate Z’-factor[4]
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.
Principles
UV-vis spectroscopy
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. [6]
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. [7] 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[8], 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.
Test
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. [9]
Reference
[1] Assay Platform for Clinically Relevant Metallo-beta-lactamases. Sander S. van Berkel, Jürgen Brem, Anna
M.
Rydzik, Ramya Salimraj, Ricky Cain, Anil Verma, Raymond J. Owens, Colin W. G. Fishwick, James Spencer, and
Christopher J. Schofield. Journal of Medicinal Chemistry 2013 56 (17), 6945-6953
[2] Xiana Qian, Shuangzhan Zhang, Shuyuan Xue, Wuyu Mao, Minqiu Xu, Weipan Xu, Hexin Xie. A
carbapenem-based
fluorescence assay for the screening of metallo-beta-lactamase inhibitors. Bioorganic & Medicinal Chemistry
Letters. Volume 29, Issue 2, 2019:322-325.
[3] Sun, & Tung-Tien. (2004). Opinion: excessive trust in authorities and its influence on experimental
design. Nature Reviews Molecular Cell Biology, 5(7), 577-581.
[4] Zhang, J.-H., Chung, T. D. Y., & Oldenburg, K. R. (1999). A Simple Statistical Parameter for Use in
Evaluation and Validation of High Throughput Screening Assays. Journal of Biomolecular Screening, 4(2),
67–73.
[5] Wang Si-wen, Chen Xiang-dong, Wang Hui et al. Establishment of high-throughput screening model for
β-lactamase inhibitor. Chinese Journal of Antibiotics. 2013, 38(2):102-105.
[6] Ke-Wu Yang, Yajun Zhou, Ying Ge and Yuejuan Zhang, Real-time activity monitoring of New Delhi
metallo-b-lactamase-1 in living bacterial cells by UV-Vis spectroscopy,Chem. Commun., 2017, 53, 8014--8017
[7] T.Li,Q.Wang,F.Chen,X.Li,S.Luo,H.Fang,D.Wang,Z.Li,X.Hou and H. Wang, PLoS One, 2013, 8, e61914.
[8] J. Ma, S. McLeod, K. MacCormack, S. Sriram, N. Gao, A. L. Breeze and J. Hu, Angew. Chem., Int. Ed.,
2014, 53, 2130.
[9] C. Dalvit, E. Ardini, G. P. Fogliatto, N. Mongelli and M. Veronesi, Drug Discov. Today, 2004, 9,
595−602.
CONTACT US
EMAIL
tju2019igem@163.com
ADDRESS
No.92 Weijin Road, Nankai District, Tianjin, China