Team:ASTWS-China/Model

• Home
• Project▾
  • Description
  • Design
  • Applied Design
• Experiment▾
  • Results
  • Demonstrate
  • Improve
  • Notebook
  • Protocol
  • Safety
• Measurement
• Human Practice▾
  • Human Practices
  • Public Engagement
  • Collaborations
• Parts
• Model
• Team▾
  • Team Members
  • Attributions

Modeling the Mec Detective System

---

Introduction

As antibiotics has been being used more and more for medical purpose, it is much more possible that water could be contaminated by antibiotics which would lead to threaten the people’s health. To tackle the problem, The Mec Detective System was designed to test whether the water is contaminated byβ-lactam antibiotics such as ampicillin, cephalothin and cefoxitin. With the Mec Detective System, the OD(optical density) values and RFP(red fluorescence protein) values can be measured. Based on the OD values and RFP values, the Mec Detective System was modeled and its effects was verified.

Data Explanation

All data used in this model is appended in the end from appendix.1 to appendix.5.

Appendix.1 lists preliminary experimental data which includes the OD values and RFP values for 0.5ug/ml ampicillin, 05ug/ml cephalothin, 0.5ug/ml cefoxitin, water at the culture time of 0hours, 2hours, 4hours, 6hours, 22hours and that of the corresponding negative control group and positive control group.

Appendix.2 lists the RFP/OD values for three β- lactam antibiotics and water based on the data in appendix.1. There are four tables and each table contains the data for one antibiotics or water. In the tables, the column ‘A’ denotes average and the column ‘STDEV’ denotes standard deviation.

Appendix.3 lists the OD values, RFP values, and RFP/OD values of different concentration ampicillin cultured overnight.

Appendix.4 lists the results of analyzing the RFP/OD values for each antibiotics and water using one-factor analysis of variance.

Appendix.5 lists the RFP/OD values at the time of 6 hour and their analysis results by one-factor analysis of variance.

Assumption

Assumption-1：All experiments are fully reacted , the concentration of solutions in test tubes are well-distributed, samples are typical.

Assumption-2：All experimental operation procedures are abiding by specifications, the data sampled are authentic and reliable.

Preliminaries

Before we conduct experiments with Mec Detective System, we should first discuss the function of Mec Detective System and phase out some assumption. As a detective apparatus for β-lactam antibiotics, it should at first decide whether the solution being detected is contaminated by antibiotics, then differentiate which antibiotics, and lastly evaluate the concentration of the antibiotics.

Because OD denotes the concentration of solution and RFP denotes the degree of effect produced by Mec system on solution, the RFP/OD means red fluorescence for single unit concentration. In the project, the RFP/OD values are the basis of modeling. Using RFP/OD value as independent variable, the four functions was defined as follows:

(1) Positive or Negative Function: Given the RFP/OD value of some solution, the function can decide whether the solution is contaminated(positive) or not contaminated(negative)

(2) Culture Time Function: Given one of β-lactam antibiotics, output the best time taken to culture by the Mec Detective System

ct(ampicillin|cephalothin|cefoxitin│water)=x (hours),x is a number in [0-24] ②

(3) Differentiation Function: Given the RFP/OD value of some solution which is contaminated by some antibiotics, the function can differentiate which β-lactam antibiotics is being contained in the solution .

(4) Concentration Function: Given the RFP/OD value of some solution which is contaminated by one of β-lactam antibiotics, the function can qualitatively analyze the concentration of the solution.

Test and Model

Then we tested the effect when β- lactam antibiotics existed. Ampicillin were added into 0.8 mL LB broth with a gradient final concentrations, samples were collected after culturing in 37℃overnight. As shown in figure 8, we can obviously see the red fluorescence of single unit of optical density(RFP/OD) increased with the concentration of ampicilin going up before reaching inhibited concentration. The Mec system can effectively detect whether the water is contaminated by AMP and analyze the concentration qualitatively when the concentration of AMP is between 0.5ug/ml and 10ug/ml. The consequences of Figure 9 also support the results by the color change.

Figure 8, the ratio of the red fluorescent intensity over OD600 under different concentrations of Amp.

Figure 9, Compared the efficiency of Mec detection system of the control group and the experimental group.

To further verify the effects of the Mec detection system towards β-lactam antibiotics, we tried experiments on three different β-lactam antibiotics: ampicillin, cephalothin, cefoxitin with the same concentration 0.5ug/ml in different culture time. The water without antibiotics was used as a control group.

By using the method of one-factor analysis of variance, we analyzed each group of data on ampicillin, cephalothin, cefoxitin, water and checked whether it is valid for each experiment. In the table1, for each group the ‘F’ is far less than ‘F crit’ and ‘P-value’ is much more than 0.05 which means the three experiments in each group are reliable and the validities of every experiment in each group are verified.

Table1, the analysis of each group of experimental data using one-factor analysis of variance

solution	SS	df	MS	F	P-value	F crit
ampicillin	89256.03	2	44628.02	0.008708	0.991336	3.885294
cephalothin	246993	2	123496.5	0.119741	0.888197	3.885294
cefoxitin	27317.8	2	13658.9	0.0106	0.989466	3.885294
water	112257.8	2	56128.88	0.123411	0.885008	3.885294

Then the curves of RFP/OD with each antibiotics in three samples was plotted comparing to the curve of water. Here the RFP/OD values of water are averaged on three samples. Figure 10a)b)c) respectively showed that with the extension of culture time, the RFP/OD of mCherry in every antibiotics test group gradually increased, which indicates that the expression level of mCherry in the medium gradually increased. Also, it was found that the RFP/OD are largely higher than that of water which manifests the Mec System can easily detect whether the water is contaminated byβ-lactam antibiotics if the culture time is more than 6 hours. Therefore we can obtain the ct function as follows:

ct(ampicillin)=ct(cephalothin)=ct(cefoxitin)=ct(water) = 6 hours ⑤

Figure 10 The RFP/OD values of β-lactams comparing to that of water

From the above, it is clearly indicated that the Mec System has good effects on detecting the β-lactam antibiotics with the culture time of 6 hours. However, the detective sensitivities of three different antibiotics are variant. Figure11 shows three RFD/OD values for each antibiotics and water at the time of 6 hour. It can be seen that the RFD/OD values of each antibiotics are distinctly higher than that of water and the detective effect for ampicillin is best, the second is cephalothin, the third is cefoxitin. The variance analysis also verified the result. Table 2 shows the ‘F’ is far greater than ‘F crit’ and ‘P-value’ is far less than 0.05 which means the Mec System has distinctively different effects on ampicillin, cephalothin, cefoxitin and water.

Table 2 The variance analysis for RFP/OD values of four solutions at the time of 6 hour

source of variance	SS	df	MS	F	P-value	F crit
interblock	24473597	3	8157866	513.8124	1.75E-09	4.066181
innerblock	127017	8	15877.13

Figure 11, the RFP/OD values of three antibiotics and water at the time of 6 hour

Based on the above analysis, the positive function and the differentiation function can be written in details as follows:

Conclusion

In brief, the Mec System can effectively detect the three different β-lactam antibiotics: ampicillin, cephalothin, cefoxitin in contaminated water with the culture time of 6 hours and qualitatively analyze their concentration. The Mec Detective System can be modeled with the following four functions:

However, with existing data we can’t deduce what the concentration is through the concentration function. We should further do more experiments on all three β-lactam antibiotics with different concentration at the culture time of 6 hours. On next step, we will enhance the Mec System so that it can detect the low concentration antibiotics sensitively and also quantitatively compute the concentration of antibiotics.

Reference

[1] Altland R B H W . Regression Analysis: Statistical Modeling of a Response Variableby Rudolf J. Freund; William J. Wilson[J]. Technometrics, 1999, 41(4):368.

[2] Cortina J M , Nouri H . Effect Size for Anova Designs[J]. Sage Pubn Inc, 1999.

[3] Clark R W . How to Use Excel in Analytical Chemistry and in General Scientific Data Analysis. By Robert de Levie. Cambridge University Press,2002.

Appendix.1 Preliminary Experimental Data

Appendix.2 RFP/OD values

Appendix.3 The experimental data for ampicillin with different culture time

Appendix.4 Analyzing the RFP/OD values for each antibiotics and water using one-factor analysis of variance

Appendix.5 The RFP/OD values at the time of 6 hour and their analysis