Part:BBa_K515102:Experience
Characterisation
This part (BBa K515102) has been characterised qualitatively and quantitatively for chemotactic response towards L (-) malic acid. The tests were performed across a range of different attractant concentrations to observe optimal functioning of the part. Qualitative tests were performed using semi solid agar assays, quantitative analysis was performed using high - throughput capillary assay with cell count for each capillary evaluated using FACS. Behavioral analysis of E. coli expressing this part was done using Wide - field microscopy, and manual tracking was used to evaluate tactic response. Negative control used was E. coli 5α strain with a selectable marker for ampicillin and kanamycin. As positive control chemotactic response of E. coli 5α strain to attractant L (-) serine was tested, as serine is compatible with endogenous chemotaxis system of E. coli 5α strain.
Qualitative assay
Two tests were performed to observe chemotactic response over a large range of chemoattractant concentration and to observe differences in much finer milimolar ranges of attractant concentration.Images of semi - solid agar plates were obtained using LAS 3000 Imager.
Table 1: Ranges of attractant concentration tested.
|
Attractant concentration |
||||||
|
Test 1 |
0 M |
10-5 M |
10-4 M |
10-3 M |
10-2 M |
10-1 M |
|
Test 2 |
0 mM |
5 mM |
10 mM |
15 mM |
20 mM |
25 mM |
Method
Bacteria were induced for chemotaxis, and filter paper circle was soaked in the bacterial culture. 20µl of attractant diluteed in the motility buffer was placed on another filter paper and positioned exactly 2 cm away from the filter paper with bacterial culture. For control only motility buffer was added to the filter paper. The plates were left overnight for bacteria to grow and move.
Test 1
Figure 1: Negative control pictured for the attraction E. coli cells, with added attractant malate. Rising consentrations of malate were tested, 0 M, 10-5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M. Circular colonies are observed with rising concentrations.
Figure 3: Positive control pictured for the attraction E. coli cells, with added attractant serine, which is compatible with native E. coli chemoreceptors. Rising consentrations of serine were tested, 0 M, 10-5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M. Circular colonies are observed with 0 M & 10-5 M concentrations, result for 10 -4 M has been rendered void due to mishandling with semi - solid agar. Possible elipse shape of the 10 -3 M sample can be observed. Directed colony shape towards the attractant source is observed at 10 -2 M, and circular colony shape is observed in the 10 -1 M sample.
Figure 7: Modified 5α E. coli cells with PA2652 receptor on pSB1C3 plasmid pictured with added attractant malate. Rising consentrations of malate were tested, 0 M, 10-5 M, 10 -4 M, 10 -3 M, 10 -2 M, 10 -1 M. Circular colony can be observed for control, elipse shape of colonies can be observed at 10-5 & 10 -3M concentrations. Elipse shaped colony can also be observed at 10 -4 M however a large spread of bacteria can also be observed to the right side of the plate. Bacteria exposed to 10 -2 M, 10 -1 M concentrations, appear as circular colonies, however the colony spread is smaller it is possible due to saturation by malate at high concentrations.
Test 2
Figure 2: Negative control pictured for the attraction E. coli cells, with added attractant malate. Rising consentrations of malate in milimolar range were tested, 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM. Circular colonies are observed with rising concentrations.
Figure 4: Positive control pictured for the attraction E. coli cells, with added attractant serine, which is compatible with native E. coli chemoreceptors. Rising consentrations of serine in milimolar range were tested, 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM. Circular colony can be observed at 0 mM concentration as control, at all other milimolar concentrations directed colony shape towards attractant source can be observed.
Figure 8:Modified 5α E. coli cells with PA2652 receptor on pSB1C3 plasmid pictured with added attractant malate. Rising concentrations of malate in milimolar range were tested, 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM. Circular colonies can be observed for 0 mM, 10 mM, 15 mM, 25 mM concentrations. Colonies exposed to 5 mM and 20 mM concentration of attractant were rendered void due to mishandling with semi - solid agar.
Comments
Semi - solid agar assay is only a qualitative measure of evaluating chemotactic movement. The only conclusion that could be drawn from the results is if bacteria do or do not perform chemotaxis towards the source. As a method for characterisation of part BBa_K515102 and its functioning within cells is very inaccurate and therefore this assay was done only to give initial clues about the behaviour of the BBa_K515102 in cells, however to analyse the parameters of the part quantitative assays were performed. Another factor of this assay is semi - solid agar which can not be replicated to exactly same measure for each sample and therefore it can to lesser or greater extent affect the assay. Some semi - solid agar plates due to fluid nature can be rendered void while manipulating with them, and the results from these have to be ignored.
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