Building devices

During this summer we tried to build a lot of devices (17 exactly) but we finally realized that they were too many for our first time. We had many problems assembling parts and checking the constructions: colony PCR did not work (or it just worked sometimes), digestion enzymes did not cut always, we could hardly obtain new parts by PCR or site-directed mutagenesis products, etc. We think most problems were caused because of the lab conditions. We worked in a practices lab and the material we used was not the best. When we noticed that the Jamboree date was too close, we had to focus in some devices and forget assembling others. Prioritizing some devices helped us to finish proposed devices. Our work moved forward faster.

You can see below the devices we finally built and what they are for.

  • Device 6. Quantify induction of PfecA promoter by GFP fluorescence.
  • Device 6 of Bacterial Crowding project
  • Device 8. Production of chemoattractant aspartate mediated by signal transduction Circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).
  • Device 8 of Bacterial Crowding project
  • Device 9. Constitutive production of chemoattractant aspartate.
  • Device 9 of Bacterial Crowding project
  • Device 14. Production of chemoattractant salicylate (for P. putida) mediated by signal transduction circuit 2 in response to ferric citrate (because we could not assemble Circuit 3).
Device 14 of Bacterial Crowding project

As you can see, we could not assemble any device related to the Sensing part of the project. Until the last day we tried to assemble the device that codes FecA/PrhA outer membrane hybrid protein (Device 17) but we did not achieve it. It seemed that this protein could have some harmful effects in bacteria. We tried to express FecA/PrhA under the control of a middle-strong constitutive promoter. First we did that using a high copy vector, but we soon changed it to a low copy plasmid. Nevertheless we never obtained colonies that harvest the hybrid protein and could survive. Two days before Wiki freezing we looked up any inducible expression vector we could use instead of our constitutive promoter. We found TetR repressed POP/RIPS generator, which showed suitable features, but we did not have enough time to test it before the Jamboree date. Anyhow, we thought that a good way to solve outer membrane protein expression problems is to use inducible promoters.

Device 17 of Bacterial Crowding project

Chemotaxis assays

The main way to test our project was to use chemotaxis assays. Over August we started to read as much articles about chemotaxis assays as we found. We soaked of these processes and tested some of them. In Assay site you can read more about all the different kinds of experiments we performed. Also we modified some of these assays.

When we started we used three bacterial strains: E. coli K-12, Pseudomonas sp. and Pseudomonas putida G7; and three chemoattractans, glutamate, aspartate and salycilate. Little by little we narrowed the possibilities to E. coli K-12 as strain and aspartate as attractant, because of its high expected chemotactic response. However, we could not achieve good results in our chemotaxis assays using tip boxes as chemotaxis chambers and needles instead of capillaries. So we changed media conditions, needle thickness, we did different dilutions… but successful results did not arrive. Finally we realized the chance involved in our misfortune, our E. coli strain got a mutation which did not allow it to move or sense chemotaxis stimulus. We checked it by using a soft agarose plate assay.

Then we changed our strain for other known motile E. coli RP437 strain which came from Sandy Parkinson lab (chemotaxis researcher). Using the new strain we performed lower scale assays (1μl capillaries, no more that 1ml of bacterial suspensions), optimized some conditions and we achieved our goal. By microscopy and dilution and plating the chemotactic response of the E. coli towards aspartate was characterized.

Here some pictures of a capillary assay of E. coli chemotaxis toward aspartate are shown. As you can see while time passes more bacteria accumulate inside the capillary and around it. This fact clearly supports the chemotaxis response of E. coli.

Capillary assay Pictures

Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.

In the last chemotaxis assay we performed before Wiki freezing we used as chemotaxis chamber a flow-chamber with three channels and 1μl glass capillaries. In both edges of each channel there was a capillary, one with aspartate and other filled with buffer. We carried out experiments with two chambers, one was incubated at room temperature during 1h and the other at 30ºC. After incubation capillaries were cleaned with water and their contents were diluted and spread in agar plates. Then plates were incubated overnight at 37ºC and counted. Results of this assay are shown below. The response to aspartate is much higher than to buffer and also the standard deviation is lower when the temperature is fixed at 30ºC.

Bacterial Crowding Chemotaxis Assay Results

Producing bacteria

The Bacterial Crowding project included sensing non-diffusible signals and producing a chemoattractant as response. In spite of the fact that we could not work with the sensing part of the project we wanted to test if our modified bacteria were able to produce enough chemoattractant to induce chemotactic behaviour in their partners. Although we had two different built chemoattractant production circuits, for salycilate and aspartate, we focused on the generation of aspartate to attract E. coli, due to its higher chemotactic response. Anyway, we started to do assays with supernatant quite late and results were not available to upload to the Wiki page before Jamboree date.

We designed several growing media in which the expression of the plasmids could be analyzed, mainly media to induce PfecA promoter. We set up inocula in these media and when they were saturated, we centrifugated and reject the pellets. The supernatants would be used in chemotaxis assays.

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