1. Summary

To accomplish our system, SMART E.coli, it is required that we make worker cells assemble to guider cells. For this we utilize a property known as chemotaxis. Chemotaxis occurs in bacteria including Escherichia coli. They are believed to be attracted toward certain substances, including L-aspartate (L-Asp). We have tried to make E. coli attract other bacteria toward itself by a substrate-stimulated production of L-Asp. When guider cells synthesize L-Asp, worker cells assemble around the guider cells and workers are able to perform their assigned task effectively. Also, we characterized the chemoattraction of E.coli toward L-Asp, and compared the results to a computational simulation. We obtained supportive evidence for the agreement of wet and dry experiments. We propose that, if we get an E. coli secreting enough level of L-Asp, we can devise an inducible cell mustering system.

1.1. The production of L-Asp

AspA synthesizes L-Asp from fumaric acid and ammonia

In previous studies accomplished L-Asp over-production, the amount of L-Asp was measured by HPLC ^[1][2]. However, having no available HPLC-apparaturs, we were unable to use this method, so we tried to detect it in alternative ways.

We first tried to make E. coli producing L-Asp using the lac promoter, BBa_R0011. WT cells transformed with BBa_K518004(lacP-RBS-AspA-d.Ter) were pre-cultured with 1mM IPTG. The culture was soaked in fumaric acid solution containing annmonia. Note that AspA synthesizes L-Asp from fumaric acid and ammonia. The reaction mix was incubated at 37 degrees celcius for 1 hour. After the incubation, the concentration of L-Asp was measured by ninhydrin staining and ultraviolet-visible spectroscopy. Unfortunately, we were not able to obtain data that was obvious. We had ninhydrin react with L-Asp produced by AspA. However, Ninhydrin probably reacted not only with L-Asp but also with the remaining ammonia.

The second attempt was TLC. Two microliters of the supernatant of the incubated reaction solution was spotted onto a TLC sillica plate, and extracted with 70% ethanol. We had expected L-Asp and ammonia to be separated. However, it was impractical to separate them using ethanol. We then tried various concentrations of acetone as a developing solvent, only to observe lanes with smears.

One of the main reasons of the failure is that our methods relied on the ninhydrin reaction. Ninhydrin certainly reacts with L-Asp. However, ninhydrin also reacts with an indispensable substrate of the AspA reaction. We should have selected a way in which only one of the reactants and products can be detected. Now then, the sequencing result shows that Assembling BBa_K518004 was successful. So, it may be possible confirm the result if the right method is selected. For example, L-Asp can be detected by HPLC and the AspA protein can be detected instead of L-Asp.

1.2. The characterization of L-Asp chemotaxis

Next, to show that E. coli moves in the direction of a higher L-Asp concentration, we carried out swarming assays. A WT colony was innoculated into a 0.25% agar LB plate and L-Asp solution was instilled. Plates were left at room temperature. After 20 hours, these plates were photographed. The representative photograph of a swarming colony is shown in Fig. 1A.

To determine the movement of colonies toward the location of L-Asp instillation, we then performed an image analysis. Obtained images were subjected to computational processing to find edges of the colonies. The processed image is shown in Fig. 1B.

Fig. 1A: A swarming colony after 20-hour incubation. A cross "×" in the plate center was the position that L-ASp was instilled. Fig. 1B: A colony image processed to find the edge. Blue line represents the border of the colony.

In this assay, we defined as a vector the distance a colony moves from the center of a colony to the initial tip position. The distances are represented in Fig. 1C. The migration distance of colonies were measured to be 38(±9) mm for a plate with L-Asp solution, while this was 13(±6) mm for a plate without L-Asp. The results clearly show that colonies exposed to the L-Asp solution swarmed significantly, compared to colonies of the control group. Data was averaged from more than 6 experiments.

Fig. 1C: movemant of colonies with L-Asp sol.(Asp+) and without L-Asp sol.(Asp-).

References

TBD

3. Summary

We characterized ultraviolet (UV)-induced promoters to devise a "UV Switch". Our SMART System requires a promoter induction at the location of substrate, so is applicable only to those substrates for which responsive promoters are already known. To address this issue, we tried to provide a well-characterized easy-inducible promoter as a genetic switch. We chose UV as an example of induction, and characterized some UV-induced promoters recAp (BBa_J22106), recNp (BBa_K518011), and sulAp (BBa_K518010). To quantitatively and accurately evaluate the promoter activity, dual luciferase assay method was employed. Our results show that sulAp works as an acute switch. Combining the UV Switch with our SMART System, it becomes possible to "switch on" the self-assembling and localization at the location of our interest.

3.1. Dual Luciferase Assay System

It is very important to be able to regulate gene expression as you like in biotechnology. To apply a regulator, such as a promoter, of the relevant strength to a gene, it is first essential to take an accurate measurement of the activity of regulators. However, it is difficult to measure the activity of regulators because the activity often changes depending on the conditions. In order to deal with this problem, Kelly et. al. (2009) proposed a BioBrick promoter assay system based on relative promoter strengths.^[3] Their system measures the fluorescence intensity of GFP placed downstream of the promoter of interest, which is used to compute the absolute promoter strength. This value is normalized to the strength of a “standard” promoter which is simultaneously measured so that differences between experiments could be minimized.

In this project, we suggest an alternative promoter assay system. In the dual luciferase assay(Hannah et al 1998), the luminescent intensity caused by firefly luciferase, used as a reporter of the promoter of choice, is compared to that caused by renilla luciferase, used as a reporter for a control promoter, both placed within the same plasmid.^[4] Importantly, because the promoter to be measured and its comparison are placed within the same plasmid, differences in expression between cells are minimized. In addition, the need to normalize cell densities between sample and control is abolished, which can often be a difficult and inaccurate task. Also, the use of luciferase as a reporter allows a quick and accurate measurement and in addition is not fluorescence-based, nullifying the need to worry about fluorescence quenching.

We assembled the Dual luciferase assay system from existing BioBrick parts, and used BBa_J23118 as the control promoter. We measure the relative intensity of this promoter to BBa_J23119, which has been already measured by the aforementioned assay, and so the intensities of promoters from the two systems can be compared to each other using BBa_J23119 as a standard.

In order to confirm that our system is workable, we assayed using our system how the activity of the lactose promoter (BBa_R0010) , an IPTG-inducible promoter, changes according to the strength of induction (Fig. 3A)

Fig. 3A

As Fig. 3A shows, it was observed that the relative activity of lactose promoter increased with increasing IPTG concentrations. The result that concentration-dependent activity was observed with little variation between trials confirms that our “Dual luciferase assay” system can be used to measure quantitatively the intensities of promoters.

3.2. Evaluation of UV-induced Promoters

As our dual luciferase assay kit (K518002) has been confirmed to work, we then assayed sulAp (BBa_K518010), which is known to belong SOS promoters, activity. (Fig. 3B)

Fig. 3B

Fig. 3B shows that the activity of sulAp in recA (+) strain on medium 4mm deep was induced after a minute exposure of UV (15W / 254nm). The relative activity of sulAp was decreased after 10 minutes exposure of UV of same strength compared with that after 1 minutes exposure of UV. It suggests that many E.coli died because of 10 minutes exposure of UV. In fact, the OD of E.coli exposured by UV for 10 minites was less than that of E.coli exposured by UV for 1 minute at recovery culture (data not shown).

Detailed explanation of our system can be shown in Data section, and full method can be shown in Method section.

We assayed promoters this time, but the “Dual luciferase assay” system makes it possible to measure not only transcriptional but also translational activity with appropriate control.

References