Team:UT-Tokyo/Project/Results

From 2011.igem.org

(Difference between revisions)
Line 60: Line 60:
:To determine the movement of colonies toward the location of L-Asp instillation, we then performed an image analsis. Obtained images were undergone a computational processing to find an edge of the colonies. The processed image is shown in Fig. 1B.  
:To determine the movement of colonies toward the location of L-Asp instillation, we then performed an image analsis. Obtained images were undergone a computational processing to find an edge of the colonies. The processed image is shown in Fig. 1B.  
<html><center>
<html><center>
-
<img src="http://2011.igem.org/wiki/images/b/bf/Ut_assay_asp_taxis.png" align="center" width = "300"><br>
+
<img src="http://2011.igem.org/wiki/images/8/82/UT_Asp_assay.png" align="center"><br>
-
Fig. 1A: A swarming colony after 20-hour incubating
+
Fig. 1A: A swarming colony after 20-hour incubatingFig. 1B: A colony image precessed to find the edge.
-
<!--fig.1A-->
+
-
<br><br>
+
-
<img src="http://2011.igem.org/wiki/images/3/33/Ut_Asp%28-%29_65h_3.png" align="center" width = "300"><br>
+
-
Fig. 1B: A colony image precessed to find the edge.
+
-
<!--fig.1B-->
+
</center></html>
</center></html>

Revision as of 09:56, 5 October 2011

Results

This page is composed of the results of our experiments.
The experiments are to evaluate the possibility and potential of our idea and can be divided into two sections: chemoattraction and production of L-Asp, and motility regulation by CheZ. Each page of the two sections includes the goal and methodological outline of experiments carried out as well as the results.
In addition, we deviced “dual luciferase assay kit” in order to establish better measurement of gene expression. This kit is designed so that luciferase assay can easily be conducted for iGEM-standardized promoters. Explanation and analysis of this kit is given here.
Details of methods and experimental conditions for each experiment are provided in Method page

Section 1: Substrate-induced Cell Assembling

1. Summary

Bacteria including Escherichia coli has a property known as chemotaxis. They are believed to be attracted toward certain substances, including L-aspartate (L-Asp). We have tryed to make an E. coli attracting other bacteria toward itself by a substrate-stimulated L-Asp production. Also, we characterized the chemoattraction of E. coli toward L-Asp, and compared the results to a computational simulation. We obtained supportive evidences for the agreement of the wet and the dry. Therefore, 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 the past studies aiming at L-Asp over-production, the amount of L-Asp was determined 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 an E. coli producing L-Asp using lac promoter BBa_R0011. WT transformed with BBa_K518004(lacP-RBS-AspA-d.Ter) was pre-cultured with 1mM IPTG. The culture was soaked in the fumaric acid solution containing annmonia. Note that AspA synthesizes L-Asp from fumaric acid and ammonia. The reaction mix was incubated at 37 degreed celcius for 1 hour. After the incubation, the concentration of L-Asp was measured through ninhydrin staining and ultraviolet-visible spectroscopy. Unfortunatelly, we could not gain obvious data. We had ninhydrin react with L-Asp produced by AspA. Ninhydrin probably reacted not only with L-Asp but also with remaining ammonia.
The second attempt was TLC. Two microlitters 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 impracible to separate them using ethanol. We then tried various concentrations of acetone as a developing solvent, only to observe smearing lanes.
One of the main reasons of the failure is that our methods relied on ninhydrin reaction. Ninhydrin certaionly reacts with L-Asp. However, ninhydrin also reacts with an indispensable substance to AspA reaction. We should have selected a way that only one of reactants and products can be detected definitely. Now then, the sequencing result shows that Assembling BBa_K518004 was success. So, it may be possible to make sure of the work if a right method is selected. For example, L-Asp is detected by HTLC and AspA protain is detected instead of L-Asp.

1.2. The characterization of L-Asp chemotaxis

Next, to show that E. coli moves in the direction of higher L-Asp concentration, we carried out swarming assays. WT colony was innoculated into 0.25% agar LB plate and L-Asp solution was instilled. Plates were left at room temperture. After further 20 hours, those plates were captured. 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 analsis. Obtained images were undergone a computational processing to find an edge of the colonies. The processed image is shown in Fig. 1B.


Fig. 1A: A swarming colony after 20-hour incubating, Fig. 1B: A colony image precessed to find the edge.


In this assay, we defined a colony movement as a vector from the center of a colony to the intial tip position. The colony movements were presented in Fig. 1C. The migration length of colonies was measured about 38(±9) mm with L-Asp solution, while 13(±6) mm 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 is obtained from more than 6 experiment.


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


-Reference-
[1]. Chao, Y. P., Lai, Z. J., Chen, P., & Chern, J. T. (1999). Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12. Biotechnol Prog, 15(3), 453-458.
[2]. Chao, Y., Lo, T., & Luo, N. (2000). Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme Microb Technol, 27(1-2), 19-25.


Section 2

TBD


Section 3

TBD