Collaboration

We are glad to cooperate with HKUST-Hong_Kong onmodeling the hypothetical behavior of bcr gene induced by lac or T7. bcr is amembrane protein that actively pumps out a variety of substrates from the cell,notably antibiotics such as tetracycline and kanamycin. Hence, expression ofbcr leads to antibiotic resistance. The collaboration was established becauseof the similarity between halorhodopsin and bcr. Both proteins are membranetransporters, and we are familiar with such scenario.

Minimum Inhibitory Concentration (MIC) is acommonly-used indicator to measure the vulnerability of bacteria in presence ofantibiotics. It is usually determined by the minimum concentration ofantibiotics that could cause observable reduction in turbidity and/or opticaldensity for an overnight LB culture. We modeled the relationship between thechange of MIC of tetracycline and kanamycin respectively and the concentrationof IPTG, which induces bcr downstream of P_lac in E. coli. The change of MIC is reported asthe ratio of the MIC of bcr-transformed E.coli over that of wildtype E. coli.

Modelingresults

Fig. 1 lac promoter activity. Curve is fit by one-site total binding model from experimental data [3], [4].	Fig. 2 Activity of native promoter [1] of bcr (acrAp+acrBp) vs. change in MIC. Curve is fit by one-site total binding model from experimental data [2], [5].
Fig. 3 Change in MIC vs. IPTG (lac promoter construct). Blue line represents tetracycline and red line represents kanamycin.	Fig. 4 Change in MIC (effect of using T7 promoter). Data are obtained from [6] and using one-site total binding model for curve fitting.

We modeled HKUST’s data with three assumptions.Firstly, antibiotics are applied during exponential phase or stationary phase.Secondly, P_lac only affects the expression of target genes (bcr).Finally, IPTG doesn’t affect other metabolic pathways except P_lacinduction. We modeled the activity of P_lac under different IPTG concentrations(Fig. 1) and the relationship between the activity of native promoter of bcrand the MIC change (Fig. 2). Then we achieved our goal to show the effect ofIPTG concentration on the change of MIC by combining the P_lacactivity and the MIC change of wild type bcr (Fig. 3). We discovered that onlya small amount of IPTG is essential to induce bcr and trigger similarantibiotic resistance compared with wild type. However, further increase ofIPTG concentration has limited effect on strengthening antibiotic resistance.This is probably due to the saturation of IPTG in bcr induction where the bcramount reaches the peak, while the concentration of antibiotics keepsincreasing.

References

[1] RegulonDB

http://regulondb.ccg.unam.mx/operon?term=ECK120014773&format=jsp#myReferences

[2] Increased expression of the multidrug effluxgenes acrAB occurs during slow growth of Escherichia coli FEMS MicrobiologyLetters Volume 207, Issue 1, pages 91–95, January 2002

[3] http://www.expressys.com/main_applications.html

[4] http://partsregistry.org/Part:BBa_R0010:Experience

[5] Analysis of a Complete Library of Putative DrugTransporter Genes in Escherichia coli J Bacteriol. 2001 October; 183(20):5803–5812.

[6] https://2011.igem.org/Team:TU_Munich/lab/results

Biobricks construct

1.     Halorhodopsin(BBa_K559000)

2.     Halorhodopsinunder T7 promoter (BBa_K590001)

3.     Halorhodopsincomplete system (BBa_559010)