Difference between revisions of "Team:Technion Israel/Color"
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<p class="text-justify"> | <p class="text-justify"> | ||
The sample was then tested for chemotaxis ability using | The sample was then tested for chemotaxis ability using | ||
| − | <a https://2016.igem.org/Team:Technion_Israel/Experiments">swarming assay</a> (Fig 4). A halo was formed after | + | <a href=https://2016.igem.org/Team:Technion_Israel/Experiments">swarming assay</a> (Fig 4). A halo was formed after |
8 hours indicating functional chemotaxis response. For our | 8 hours indicating functional chemotaxis response. For our | ||
| − | <a https://2016.igem.org/Team:Technion_Israel/Experiments">chip assay</a> more instance color is needed, in order to obtain | + | <a href=https://2016.igem.org/Team:Technion_Israel/Experiments">chip assay</a> more instance color is needed, in order to obtain |
that the sample was centrifuge and resuspend in a smaller volume | that the sample was centrifuge and resuspend in a smaller volume | ||
of TB medium, increasing the bacterial concentration by 10 folds. | of TB medium, increasing the bacterial concentration by 10 folds. | ||
Revision as of 19:46, 19 October 2016
Introduction
Chromogenic proteins usually serve as a useful reporter in determining gene expression levels without the need of a fluorescent microscope. However, the FlashLab technology implements these chromogenic proteins for a different purpose. Due to the chips structure, when the bacteria moves towards or away from substance, a cluster is formed and the presence of chromogenic proteins allows the user to spot it in the naked eye without the need for a complex device (for more information about our chip click here).
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).
Fig. 1: High expression system of chromogenic protein.
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
The first step, as mentioned in the implementation section, was to clone a strong promoter (J23100) upstream to each part, creating a high expression system. The biological system was then transformed to E.coli Top10 strain and UU1250 strain. Plating results showed colored colonies, for both strains, as expected. Colored colony from each type was incubated overnight at 37℃ LB medium. Overnight incubation resulted a medium that appeared as colored, doe to high concentration of bacteria expressing chromoproteins. After centrifuging the medium sample a colored pellet can be seen and the supernatant return to in its original color (Fig 1).
Fig. 1: E.coli Top10 strain expressing (left to right): mRFP, tsPurple, amilCP.
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
FlashLab system is based on the idea of moving bacteria expressing chromoproteins using two different expression plasmids. In order to verify the idea Tar was chosen as the chemoreceptor and amilCP as the chromoprotein. The first plasmid (K1992004) causes the expression of Tar chemoreceptor. The plasmid contains chloramphenicol (CM) resistance. The second plasmid causes the expression of chromoprotein (see Implementation – part haven’t been submitted). The plasmid contains ampicillin (Amp) resistance. The two plasmids were co-transformed to UU1250 strain. The strain was then screened by using two antibiotic LB agar plates. The plating results showed colored colonies and non-colored ones (Fig 2), that due to the non-compatible ORI of the two plasmids (see Outlook section).
Fig. 2: Co-transformtion of K1992004 and tsPurple expressing plasmid to UU1250 strain.
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).
Fig. 3: Left tube - UU1250 strain expressing Tar chemoreceptor only (K1992004). Right tube - UU1250 strain expressing Tar chemoreceptor and tsPurple chromoprotein.
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.
Fig. 4: Swarming assay results (left to right) - UU1250 expressing only Tar chemoreceptor; UU1250 expressing Tar chemoreceptor and tsPurple chromoprotein; UU1250 only; ΔZ - E.coli expressing all chemoreceptors.
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.
References:
1. MAEDA, Kayo, et al. Effect of temperature on motility and chemotaxis of Escherichia coli. Journal of bacteriology, 1976, 127.3: 1039-1046.
