Team:Amazonas-Brazil/Results
Characterization of Escherichia coli Nissle 1917
Escherichia coli Nissle 1917 (EcN) is a powerful chassis for synthetic biology and have been widely used as probiotic since its discovery in the second half of the 19th century by Alfred Nissle1. We aimed to engineer and standardize Escherichia coli Nissle 1917 for iGEM, providing tools for boolean logic operations executed through its robust machinery. In that direction, we successfully transformed Nissle by electroporation and characterized its EcN growth curve, as well as quantified the expression of GFP and RFP using standardized BioBrick parts cloned in the pSB1C3 backbone.
E. coli Nissle 1917 strains were grown overnight in Luria-Bertani (LB) containing ampicilin (100 µg/mL) at 37°C and 200 rpm. Cultures were diluted in fresh LB until achieve 0,1 OD with the corresponding antibiotic and transferred to a 96-well plate (50 µL/well). Samples were always made in triplicates and a blank of LB. During 8h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 1 hour.
Fig. 1. - RFP sinalization system in pSB1C3 running on E. coli Nissle 1917.
Characterization of AND gate
We quantified the dynamic range of our genetic circuit (BBa_K3320006) in response to different concentrations of hypoxia and lactate. To simulate different concentrations of oxygen for the hypoxia-inducible promoter (BBa_K387003) we used syringes, as you can see in Figure 2, Inspired by the protocol established by the iGEM Team McMaster II for the same promoter in the composite biobrick BBa_K2493001. The construction was transformed in EcN and inoculated in LB broth in different conditions of concentration of lactate and oxygen and then incubated at 37° C in shaker for 16 hours at 220 rpm. After the incubation, the GFP fluorescence was measured by Hidex Chameleon Microplate Reader.
Fig. 2 - Simulation of different concentrations of oxygen
In this experiment, we expected to understand the input-output relation with the goal to better design a real-world model with potential for environment recognition and drug delivery. The data is shown below.
Figure: GFP expression under control of our AND logic gate in different hypoxic and lactate conditions.
The first condition indicates the state of hypoxia, the second hypoxia and 0,1 mM of lactate and the third hypoxia and 1 mM. It is possible to conclude that there is an increase of GFP expression related to lactate increase, as indicates the fluorescence difference between samples with 0,1 and 1 mM. However, in the condition of hypoxia and absence of lactate, the system is still ON, which indicates a leaky regulation of the hypoxia-dependent promoter. We hope to continue our study to reach an AND gate behavior.
Secretion System
As a result of postal service problems, we were not able to clone all the secretion system biobricks. We hope to continue our experiments and clone this unit since this is an essential part to perform the drug delivery
Future Perspectives
a) Characterization of the current genetic construction using advanced and accurate methods such as RNAseq and flow cytometry
b) Individual characterization of each promoter from the synthetic genetic circuit
