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Exogenous Alkanes Induce
Here, we want to test the response of Alk-Sensor to exogenous alkanes.
We add alkanes outside the cell to test the function of Alk-Sensor:
Construct:
Strain 1: Palkm and AlkR downstream of J23103, are cloned into two vectors--PSB3K3 and PSB1C3, respectively, which we hope to prevent the influence of the constitutive promoter J23103 on the reporter gene RFP.
Strain 2: We construct a blank vector without AlkR as blank control, and on the other vector the reporter gene RFP is still on the downstream of the inducible promoter PalkM.
Characterization: The two strains are cultured in 3ml antibiotics （kan and cm） M9 medium for about 12 hours. Harvest the bacteria cells by centrifuge.
Result:
No. 55 is not red at all. No. 54 with water added shows a bit of red. No. 54 with alkanes added as inducers is the reddest among three. Alk-Sensor can respond to exogenous alkanes. Besides, No. 54 with water added is less red than with alkanes added because our sensor has a weaker response to water than to exogenous alkanes.
Specific detection
Characterization: The two strains are cultured in M9 medium for about 12 hours. Adjust the concentration of suspension culture until OD reaches 0.6, then take out 3mL and add 300μL Octane(C8),Decane(C10),Dodecane(C12), Tetradecane(C14), Hexadecane(C16) and Octadecyl(C18) respectively. After that, induce for 24 hours. Measure the ratio of fluorescent intensity (fluorescent intensity per OD) of cells, which is shown below:
From this diagram, we can clearly see that induced by saturated Octane(C8), ratio of fluorescent intensity is the strongest. We infer that there are two causes of the low ratio of fluorescent intensity of other alkanes.
1.Alkanes are hydroforbic molecules, which makes alkanes with carbon chain lengths equal to or larger than 10 hard to dissolve in water to induce alkR.
2.Ratio of fluorescent intensity of Dodecane(C12) induced is larger than those induced by Decane(C10), proving that alkR has a specifical detection of Dodecane(C12). This is also consistent with the reference.
Upper limits of detection
We choose Octane(C8), because it is commonly used to characterize alkane-sensor in most references, and we choose Pentadecane(C15), because our endogenous alkane producing module produce this kind of alkane.
We choose the strains of No.54 and culture them in 3mL LB medium until OD reaches 0.6, add C8 and C15 of a concentration range from100nmol/L to 1mmol/L, over-night cultured. All of them are utilized to measure fluorescent intensity of cells.
C8
C15
Result: With the increasing concentration of alkanes, ratio of fluorescent intensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, the increase of ratio of fluorescent intensity becomes smooth, approaching its upper limit at the concentration of 10μmol/L C8 in LB medium, and approaching its upper limit at the concentration of 1μmol/L C15 in LB medium.
We choose the strains of No.54 and culture them in 3mL M9 medium until OD reaches 0.6,add C8 and C15 of a concentration range from 100nmol/L to 1mmol/L, over-night cultured. All of them are utilized to measure fluorescence intensity of cells.
Result: With the increasing concentration of alkanes, ratio of fluorescent intensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, ratio of fluorescent intensity increases constantly. At the concentration of 10nmol/L to 2nmol/L of C8 and C15, stronger positive correlation is showed than higher concentration of C8 and C15.
We choose the strains of No.54 and culture them in 3mL M9 medium, until OD reaches 0.6,add C8 and C15 of a concentration range from 100nmol/L to 1mmol/L and 30uL DMSO, over-night cultured. All of them are utilized to measure fluorescent intensity of cells.
Result: With the increasing concentration of alkanes, ratio of fluorescent intensity is also increased. There is a positive correlation between concentration and ratio. Using logarithmic fitting curve, we find that with the increasing concentration of alkanes, the increase of ratio of fluorescent intensity becomes smooth, approaching its upper limit at the concentration of 10μmol/L C8 in M9 medium, and approaching its upper limit at the concentration of 1μmol/L C15 in M9 medium. At the concentration of 10nmol/L to 2nmol/L of C8 and C15, strong positive correlation is also showed.
DMSO is a polar aprotic solvent which increases permeability of cell membranes and helps alkanes to induce alkR. However, compared with DMSO-free medium, growth of cells are inhibited.
