Team:HKUST-Hong Kong/mic.html

2. MIC

Culture Tests

0. Introduction
In order to quantitatively demonstrate the effect of indole charity as well as our construct’s ability to negate it, we have decided to perform a series of minimum inhibition concentration (MIC) tests, where we subjected different strains and mixtures of E.coli to an antibiotic gradient and cultured overnight (18 hours). The OD600 readings of each test were recorded afterwards and will be shown in later sections for comparison. It is important to note that for each test, we did incubations using both 15ml Falcon tubes (2ml culture) and 1.5ml microcentrifuge tubes (1ml culture) to observe whether oxygen supply would affect the population distribution. [Top]

I. Wild Type (RR1) MIC Test

Phase 1 - Kanamycin MIC test

Experimental Design and Aim:
RR1 is a derivative of the common Escherichia coli strain K12 and is not known to have any antibiotic resistance other than for streptomycin. Hence it was arbitrarily chosen as the non-resistant ‘wild type’ for our tests. A simple MIC test was conducted for RR1 to serve as a benchmark for comparison with later experiments; and kanamycin, an aminoglycoside, was opted as the antibiotic of choice. This was primarily for two reasons:
First, the kanamycin resistance gene incorporated into our selection plasmids functions through producing a mutated ribosome that is insensitive to kanamycin. Unlike some other forms of resistance where antibiotic molecules are directly inactivated, this method ensures that the antibiotic levels remain relatively constant throughout the experiment, as well as prevents the appearance of satellite colonies during plating.
The other reason is because kanamycin can be both bacteriostatic and bactericidal, depending on its concentration and the microbe’s resistance. As our experiments involve plating out cultures for colony counting, it is useful to have a clear differentiation between cells severely affected by kanamycin (bactericidal effect kicks in and removes vulnerable cells) and those that are sustained by indole (cells either kept in stasis or are unaffected, and thus will have colonies). This allows us to better observe the potency of indole charity when we apply kanamycin at below-working concentrations.

Results:
The MIC of RR1 was found to lie between 6~9µg/ml.

Phase 2 - Kanamycin MIC test with indole supplement

Experimental Design and Aim:
Indole has been proposed as a key signalling molecule produced by unstressed (high resistant) E. coli as a form of ‘charity’ that grants stressed (low resistance) cells passive immunity against antibiotics. This enables such stressed individuals to continue to survive and proliferate. Indole functions by inducing the expression and activity of multidrug efflux pumps to expel antibiotics and toxins, as well as activating oxidative-stress protective mechanisms to minimize DNA damage.[1] In an attempt to ascertain and quantify this effect, we repeated the kanamycin MIC test, this time supplementing the LB medium with different concentrations of indole (300µM and 1mM).

Results:
The effect of indole on the MIC for RR1 varied under different concentration. At 300µM, which was the documented natural concentration of indole maintained by unstressed E. coli [1], we saw a clear increase in MIC as shown by a shift of the curve to the right of the non-indole MIC curve. The rate of decline of OD600 (an estimation of cell concentration), also indicated that at 300µM, indole is helping RR1 survive better in kanamycin.
On the other hand, further increasing the concentration of indole to 1mM did not seem to yield higher MICs. Rather, the results indicated that RR1 performed similarly in 1mM indole and in normal LB, at times even worse. We have several possible explanations for this. First, indole is inherently toxic. It is possible that at 1mM, the toxicity of indole overcame the benefits it provided, and instead began to kill rather than protect cells. Another possibility is that over-promoted expression of passive immunity mechanisms due to higher than natural concentrations of indole over-exhausted cell resources, leading to cell senescence or even death. [Top]

II. Mixed Culture MIC Tests

Phase 1 - Wild type (RR1) with RFP-labelled kanamycin resistance strain (RFP) (99:1)

Experimental Design and Aim:
As metioned previously, when E. coli cultures are subjected to antibiotic selection pressure, a small number of naturally resistant individuals, at some cost to themselves, provide protection to other more vulnerable cells by producing indole, resulting in an overall enhancement of the survival capacity of the population in stressful environments. To mimic this naturally occurred phenomenon, a kanamycin resistant strain, which represents the mutants, was introduced into the RR-1 at 1:99 ratio. This kanamycin resistant strain was labeled with RFP for easy recognition. The ratio of kanamycin resistant strain, KanR/RFP, to RR-1 was recorded for later comparison with that of later mix culture assays.

Results:
We can clearly see the effect of the charity work from our result. Even under kanamycin concentration of 25µg/ml, which is half of the working concentration of kanamycin and almost 3 folds of RR-1 MIC, RR-1 is still growing rapidly and maintain the majority of the overnight culture. The OD600 result didn’t show a clear co-relation with kanamycin concentration and is floating around 1.1.

The column chart also shows that the ratio of RFP to RR-1 falls between ½ and ⅓ after overnight culture. This ratio may change when the kanamycin concentration approach its working concentration. We plan to prove this in our future testing.

Phase 2 - Wild type (RR1) with kanamycin resistance T4MO (GRP)

Experimental Design and Aim:
In order to interfere the indole charity work and obtain a more efficient selection, we introduce a plasmid which encodes Toluene-4-Monooxygenase (T4MO), an enzyme that catalyzes the oxidation of indole into indigo. To test its effect, we design a T4MO/KanR and RR-1 (1:1) mixture culture MIC test. In this test, we assume that the indole degradation rate of T4MO will be close to the indole producing rate of itself along with the mutated minority in RR-1, which means a lower MIC of RR-1 will be observed in comparison of the KanR/RFP and RR-1 mixculture due to the absence of the indole charity work. The ratio of T4MO/KanR to RR-1 was also kept in the testing for later comparison with that of the 3 way mix culture assay.

Results:
Since the charity work is simply being weakened by the introduced enzyme, RR-1 is still growing quite well under kanamycin concentration of 25µg/ml. However, the ratio of the two strain, T4MO/KanR and RR-1, is different from former result from mixed culture of RFP/KanR and RR-1. Under lower kanamycin concentration, RR-1 still remain to be the majority of the culture and the difference is not very obvious. When kanamycin concentration exceeds 10µg/ml, we can see that T4MO out-competed RR-1 and became the majority of the overnight culture. Comparing with the ratio got from RFP&RR-1 mixed culture, we can draw the conclusion that the charity work of is weakened and the efficiency of ( )

Phase 3 - Wild type (RR1), RFP-labelled kanR strain and GFP-Labeled KanR T4MO (98:1:1) [???]

It has been proved in the phase 2 that T4MO does interrupt the indole charity work. So in the next step, we plan to practice our model, which is introducing a T4MO strain into the environment predominantly consisting of RR-1with few kanamycin resistant mutants. By comparing the resulting ratio of RR-1 to the antibiotic resistant strain to that of the T4MO and RR-1 Mix culture, we may observe again the strong effect of indole; by comparing the resulting ratio of RR-1 to the antibiotic resistant strain to that of the KanR/RFP RR-1 Mix culture without T4MO, we would be able to tell how T4MO takes effect. [Top]

III. Conclusion

[lalaala] [Top]

IV. Future Plans
Phase II - Wild type (RR1), RFP-labeled kanR, and GFP-labeled T4MO/Bcr mix

Our ultimate goal is to boost the selection efficiency by introducing a T4MO/Bcr strain, which can interfere with the indole charity work. The Bcr gene, which encodes a multidrug pump, keep this strain survive and produce T4MO. By adjusting IPTG concentration, this strain will keep working for a certain period of time and die afterwards as to the accumulation of kanamycin inside the cell. However, as we didn’t have time to characterize the efficiency of Bcr, and another essential part of our project, the alternative selection method, is still in progress yet, we are unable to do this construction and perform further testing.

(insert a picture here showing out ideal construction) By having this strain in the population, the charity work will be restricted, so that the selection process can be done efficiently without applying over dosage of antibiotics, and the presence of this strain can be controlled by us so that this alien strain only performs the duty of degrading indole and brings no side effect on the whole selection process. [Top]

V. Biobrick construction

Bcr Bcr is a type of multidrug efflux pump, which are integral membrane proteins that utilize cellular energy to extrude antibiotics or biocides actively out of the cell. It belongs to the major facilitator superfamily (MFS), and is known to contribute to multidrug resistance in E. coli.

Under normal growth conditions, a large number of drug efflux pumps are thought to be weakly expressed. In particular, literature documents Bcr to confer varying degrees of resistance to several kinds of antibiotics when overexpressed; including bicyclomycin (selection-capable), tetracycline (8-fold MIC increase*), and kanamycin (4-fold MIC increase*).

In our iGEM project, we planned to construct a biobrick with the pLac promoter driving expression of Bcr. The reason behind this is to take advantage of the additive effect of IPTG on pLac activation. We hope that by varying the concentration of IPTG, we can control the level of expression of Bcr and thus manipulate the mutant E. coli’s MIC to certain antibiotics.

However, as the time limited,we only submit a plasmid contain only the bcr gene to part registry. You can use it for selection of bacteria in the future. [Top]

*: compared with wild type

VI. Appendix

[Extra data] Protocols will probably be included in the Notebook section [top]

[1] http://www.nature.com/nature/journal/v467/n7311/abs/nature09354.html
[2] http://www.scielo.br/pdf/gmb/v26n2/a17v26n2.pdf