Implementation

Three chromogenic proteins (chromoproteins) were tested for the S.Tar system, all which were provided and extracted from the iGEM 2016 kit. Each part contained RBS, chromoproteins encoding sequence and a double terminator. The different parts contained the next proteins:
- tsPurple, visible as purple color (K1357008).
- amilCP, visible as blue color (K1357009).
- mRFP, visible as red color and can serve also as red fluorescence protein (K1357010).

To test the expression and visibility of those proteins, a strong promoter (J23100) was cloned upstream to the RBS using the RFC10 assembly (Fig 1).

This plasmid is one of two plasmids constructing our FlashLab system, as the other is plasmid expressing a chemoreceptor. The two plasmids are co-transformed to UU1250 strain causing the expression of both, a designed receptor and a chosen color. Each plasmid contains different antibiotic resistance allowing easy screening for strain expressing both proteins.

Results

As bouth strain showed similar results the next experiments conducted only with the UU1250 strain, the strain which used for chemotaxis assays. Growth conditions for chemotaxis assays require a minimal growth medium, TB, and a temperature of 30℃(1).
Overnight growing, in this condition, of UU1250 strain expressing chromoproteins resulted colorless medium, although bacterial concentration was high. In order to isolate the factor causing this issue, two different growth conditions were set. Incubation at 37℃ in TB medium and incubation at 30℃ in LB medium. At 37℃ TB medium, color was detected. The color was less intense in compare to the 37℃ LB medium, but still high enough to detect by a naked eye. Moreover the pallet showed color with similar intensity to the 37℃ LB pellet. As for the 30℃ LB medium no color detected after overnight growth. In addition the pellet was also colorless.
Due to these results one can easily imply that the growth temperature has a significant influence on the chromoprotein expression. To achieve color intensity at the right conditions a two-stage growth was conducted. The first stage is incubation at 37℃ LB medium in order to cause a high expression of chromoproteins. The culture is then centrifuge and resuspend with TB medium. The second stage is incubation at 30℃ for 3 hours restoring chemotaxis abilities. This two-stage growth allows both color expression and chemotaxis ability to the bacteria. This two-stage growth was proven as effective in matter for chromoprotein expression, as for chemotaxis ability test, the two palsmid system was conducted.

The two plasmid system

The colored colonies were isolated and grown using the two-stage growth method mentioned previously. The result is high density and colored medium (Fig 3).

The sample was then tested for chemotaxis ability using swarming assay (Fig 4). A halo was formed after 8 hours indicating functional chemotaxis response. For our chip assay more instance color is needed, in order to obtain that the sample was centrifuge and resuspend in a smaller volume of TB medium, increasing the bacterial concentration by 10 folds. Results can be seen in the Proof of concept page.

Outlook

We succeeded to get colored bacteria grown in the optimal condition for our assay. At that point both, the chemoproteins and the receptors, cloned on high copy plasmid with the same ORI - pMB1 (pSB1C3 and pSB1A2). Usually, plasmids with the same ORIs are incompatible because they will compete for the same machinery, creating an unstable and unpredictable environment. For future plan we mean to clone one of the expression systems to plasmid containing different ORI, compatible to pMB1 ORI. This adjustment will improve the stability of our system and allow better control over the expression of each protein.