IGEM:IMPERIAL/2009/M1/Modelling/Analysis/literatureIPTG

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The E.ncapsulator: the disease killer

Contents

Summary

Effect of IPTG on pInt bearing cells and growth: (paragraph from paper)

In the absence of the inducer, pInt-bearing cells carrying a kanamycin resistance-encoding plasmid without a tRNA insert showed a faster growth rate than those with one. However, once IPTG was added the two populations of cells show a reversal in the pattern of growth. Cells with an AGA tRNA plasmid showed faster growth, whereas those without the tRNA plasmid slowed down. Cells bearing only an AGA tRNA plasmid or the control plasmid alone showed no differences in growth rate with or without the inducer (data not shown).The IPTG-induced slow growth of pInt-bearing cells without a tRNA-supplementing plasmid can be understood in the context of the already described inhibitory effects of int mRNA on protein synthesis. It may be speculated that in the presence ofthe inducer, these cells enter the stationary phase sooner because of depletion of their rare Arg tRNA. See figure 5 in the paper for the growth curve. [1]

Study of IPTG by looking at different beta-galactosidase concentrations (of enzyme)

  • Abstract:- A special strain of bacterial cells were transformed with a reconstructed plasmid carrying the

gene for β-Galactosidase enzyme in order to study its activity in response to the different concentrations of IPTG and to assess the best concentration for the maximum expression of β- Galactosidase and to identify the inhibitory concentrations.

  • Intro:- Expression of proteins is controlled and regulated by a number of regulatory molecules.Inducers are molecules which interact with the binding regions on the DNA and influence the expression of that gene. Lac Z is one of the genes which can be induced by isopropyl betathiogalactoside(IPTG); the induction however can increase or decrease the expression/activity of

the enzyme depending on the concentration of IPTG used. Assessing the activity of β-galactosidase induced by IPTG can be useful in establishing the effective concentrations which either increase or decrease the activity of the enzyme. Lac Z gene is used as a reporter gene in many experiments to indirectly assess the activity of other genes or the interactions of proteins with in the cell. Since IPTG acts as an inducer for Lac Z gene, it is important to know the effective concentration of IPTG which will lead to maximum expression of Lac Z.One such experimental model, which uses Lac Z gene as a reporter gene is the yeast two hybrid system. Expression of β-Galactosidase is used as an indirect marker for measuring the interaction of targeted proteins. A positive interaction between targeted proteins will induce a transcription of the reporter gene to quantify the intensity of the interaction. IPTG acts as an inducer in this experimental model.

  • Conclusion:The activity of β-Galactosidase enzyme varied with different concentrations of IPTG.Concentrations between 0.1 μm - 1 μm of IPTG showed decrease in expression/activity of the enzyme, maximum expression/activity was seen at 50μm concentration of IPTG from which point the expression/activity started decreasing to a point where the expression/activity was less than the basal level for concentrations above 800μm.This analysis concludes that the maximum expression/activity was seen with 50μm concentration of IPTG and concentrations beween 0.1 μm - 1 μm and greater than 800 μm had a high degree of inhibition in which the expression and activity of the enzyme was less than the basal level expression/activity. All other concentrations of IPTG had variable levels of induction and inhibition.[2]

Another study looking at IPTG growth curves

Growth rate response, cell survival and effect of toxicity on bacterial growth: After addition of IPTG, the growth of bacterial cells was monitored by measuring A600 of the culture after every 1 h interval for 7 h. In the presence of IPTG, the growth of cells decreased after 2 h and ceased after 5 h; but in the absence of IPTG, the growth continued till 6 h and then stopped (Fig. 2A). However, such inhibition of bacterial growth was not observed when the mouse ERa transactivation domain (mouse ERa-TAD) was expressed under similar conditions (data not shown). Furthermore, the number of colony forming units in induced culture containing 1 mM IPTG was significantly reduced as compared to uninduced culture without IPTG (P = 0.01, P\0.05) (Fig. 3). Also during transformation, the plate containing low number of colony in the presence of 1 mM IPTG and ampicillin reflected toxic effect on growth. However, the growth was normal in the absence of IPTG (Fig. 4). [3]

References

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  1. Error fetching PMID 8631683: [IPTG1]
  1. Effect of Variable Isopropyl β-Thiogalactoside Concentrations on β-Galactosidase Activity,Avinash M. Baktula and Genise A. Nolan, Department of Biology, Western Kentucky University [IPTG2]
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  1. Error fetching PMID 15188500: [IPTG3]
  1. Forum link [IPTG4]
Error fetching PMID 17786586:
  1. Error fetching PMID 17786586: [IPTG5]







































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