Collaboration

We are glad to cooperate with HKUST-Hong_Kong on modeling 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 of bcr leads to antibiotic resistance. The collaboration was established because of the similarity between halorhodopsin and bcr. Both proteins are membrane transporters, and we are familiar with such scenario.

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

Modeling results

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_lac induction. We modeled the activity of P_lac under different IPTG concentrations(Fig. 1) and the relationship between the activity of native promoter of bcr and the MIC change (Fig. 2). Then we achieved our goal to show the effect of IPTG concentration on the change of MIC by combining the P_lacactivity and the MIC change of wild type bcr (Fig. 3). We discovered that only a small amount of IPTG is essential to induce bcr and trigger similar antibiotic resistance compared with wild type. However, further increase of IPTG concentration has limited effect on strengthening antibiotic resistance.This is probably due to the saturation of IPTG in bcr induction where the bcr amount reaches the peak, while the concentration of antibiotics keeps increasing.

References

[1] RegulonDB

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

[2] Increased expression of the multidrug efflux genes acrAB occurs during slow growth of Escherichia coli FEMS Microbiology Letters 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 Drug Transporter 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)