Team:UPNAvarra Spain/Experiments
Experiments
1.Cloning
To develop our project, we designed a series of plasmids (see design section) with the aim to detect the presence of nitrate, cooper, mercury and cadmium in water samples.
In order to achieve our goal, we have used the BioBrick assembly method. This approach is based on specific restriction enzyme assembly. Different DNA fragments (promoters, RBS, coding sequences…) have been standardized and called as “parts” adding identical flanking restriction sites that are not contained within the sequence (known as prefix and suffix sequences). In this way, parts can be combined together and the original flanking sites re-used for the next round of assembly and finally create new BioBricks with novel properties
In general, our objective is to create composite parts with the following structure: Prefix-Promoter-RBS-CDS-Terminator-Suffix. The basic parts we used were coming directly from the iGEM distribution kit or were obtained by PCR amplification form different composite parts also present in the kit. As an example, we will explain the process that we followed to obtain the biosensor capable of detecting nitrate, considering that it was similar for the other plasmids.
We start with the digestion of the plasmid containing the nitrate sensitive promoter with EcoRI-SpeI restriction enzymes. At the same time, the plasmid containing the RBS (BBa_B0030) is linearized by EcoRI-XbaI enzymes. During ligation, the promoter is inserted into the plasmid containing the RBS, according to the complementarity of the enzymes: EcoRI-EcoRI and SpeI-XbaI. In the next step the new construction is digested with EcoRI-SpeI and ligate into the plasmid containing the blue chromoprotein digested with EcoRI-XbaI. The last step is the integration of the terminator (BBa_B0015) downstream of the chromoprotein, repeating the same process of digestion and ligation. The plasmid used as backbone in all cases was pSB1C3, carrying chloranphenicol resistance. The general workflow is explained in the following video (and detailed in the protocols section).
In order to check that the process works, colony PCR and miniprep digestions were performed to make sure that the sequences had the correct number of base pairs in each case. At the end of the process, all constructions were confirmed by sequencing.
Finally, we obtained five different plasmids ready to be transformed on E. coli cells:
Finally, we obtained five different plasmids ready to be transformed on E. coli cells:
- NITRATE-amilCP blue (BBa_K2817007, or our twin BBa_K3287000)
- NITRATE-amilGFP yellow (BBa_K3287001)
- COOPER-amilCP (BBa_K3287002)
- MERCURY-tsPurple (BBa_3287003)
- CADMIUM-efordRed (BBa_K3287004)
2.Inductions
Once we finished the constructions, we tested their ability to detect nitrate, cooper, mercury or cadmium. We performed nearly the same experiments several times to adjust the assayed concentrations and induction times.
- Potassium nitrate (KNO3): 0, 1, 2, 4, 8 and 16 mM
- Copper chloride: 0, 5, 75, 250 and 500 mg/L
- Mercury chloride (HgCl2): 0, 6, 30, 60, 120 and 240 µg/L
- Cadmium chloride: 0, 0.01, 0.1, 1, 10 and 100 µM
We leave the cultures growing under agitation for 6 hours (Nitrate and cooper) or 24 hours (Mercury and cadmium).
After those incubation times, we take 2 ml of each culture, centrifuge them, remove the supernatant and take pictures of the remaining pellets. The imaging process is extremely relevant for the conditions of the dataset used in the mathematical models. We decided to take two sets of pictures, so as to evaluate two different conditions. The first dataset was taken at the same daytime (hence, with similar daylight) on standardized conditions (camera setting, tripod, background, etc). Static lightning setup was discarded as unnecessary. This first dataset is used to prove the linear relationship between concentration and color. A secondary dataset was generated taking images at different daytimes, with different camera settings. With this dataset we intend to simulate (part or all of) the variability of images that will be gathered in a real-world application. Hence, it is used to prove that the concentration-color relationship observed in standardized images can also be observed in condition-variable images.
After that, once we proved that the variation in the color of the bacteria relates to the concentration of the heavy metal or nitrate by means of a mathematical model, we used the biosensors to test the water samples following the same protocol (instead of adding the specific substance at known concentrations, we add the sample x).
