Part:BBa_K519010:Experience

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Applications of BBa_K519010

Zinc Sequestertration (Newcastle iGEM 2016)

As it is hypothesized that smtA expression confers zinc tolerance we conducted experiments growing E. coli in growth media containing various zinc sulphate (ZnSO4) concentrations with the aim of finding the minimum inhibitory concentration. This was part of the Newcastle 2016 iGEM team's work on [http://2016.igem.org/Team:Newcastle/Parts#bio-varistor 'biological variable resistors'].

In this experiment, we used 6mM of zinc sulphate (ZnSO4) and carried out a dilution of half for each column of a 96 well plate to try and identify the minimum inhibitory concentration for the E. coli to grow in. Cells were again placed in the pSB1C3 backbone and fresh cultures were grown at 37°C overnight. The cells were diluted down to a starting optical density of around 0.05 at 600nm using LB broth with chloramphenicol. Then, 100μl of cells were placed in the wells with the corresponding ZnSO4 concentration which was diluted using LB with chloramphenicol. This experiment was left for 16 hours to give the E. coli time to grow and replicate, see figure 1.

Figure 1. Shows the minimum inhibitory concentration for E. coli growing in ZnSO4. The cells were left for 16 hours to grow in different zinc sulphate concentrations with LB broth and chlormaphenicol. OD600 was measured after this 16 hour growth period.

From this experiment, we found that the minimum inhibitory concentration was 3mM of ZnSO4 for E. coli. This concentration was then used in our second experiment to test whether our “Variable Resistor” (BBa_K1895999) construct was able to sequester zinc in the presence of arabinose.

Growth with Zinc and Arabinose

The minimum inhibitory concentration was used for this experiment to determine whether the construct would sequester the zinc if arabinose was present. For this, we used a 96 well plate and used half of the plate to contain 3mM of ZnSO4 with 0.5% of arabinose and LB broth with chloramphenicol. The other half of the plate contained the 0.5% of arabinose with LB and chloramphenicol.

The cells were grown up in 10ml of liquid culture of LB broth with chloramphenicol overnight at 37°C. The cells were then diluted down to an OD of 0.05 at 600nm and 100μl of the cells were then added to each well. The cells were then left to grow for 16 hours and the OD600 was measured every 5 minutes. The results can be seen below in figure 2.

Figure 2. Growth curves of E. coli. Those growing with zinc were grown in 3mM of ZnSO4 with 0.5% arabinose and LB broth with chloramphenicol. Those growing without zinc were grown in 0.5% arabinose and LB broth with chloramphenicol. The OD600 was measured every five minutes, and the cultures were shaken in between the readings. The cells were grown for 16 hours. VR = Variable resistor (BBa_K1895999), BBa_K1895100 = Our positive control and BBa_K1895100, WT= Wild-type.

From these results, we concluded that our ‘Variable Resistor’ (VR) construct does not grow in ZnSO4 when arabinose is present. The cells only grew when zinc was not present. This shows that the E. coli does not sequester zinc when the smtA gene is expressed.

User Reviews

UNIQb39c5ae49034a94b-partinfo-00000000-QINU

No review score entered. iGEM Nagahama_2014

We found the sequence of this part has the PstⅠ site. So we mutated C→T at 96 (BBa_K1342003)

BBa_K1620007 iGEM UFSCar-Brasil [http://2015.igem.org/Team:UFSCar-Brasil.html]

This SmtA was improved to eliminate an internal restriction site for PstI, the 98th base was changed G -> A. No residues were changed. Figure 1: Improved SmtA (BBa_K1620007) compared to BBa_K519010; this new sequence is biobrick compatible due the removal of a PstI restriction site previously present in BBa_K519010.

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