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Aim of this experiment
To verify AlkSensor’s ability to distinguish different in vivo concentrations of alkane and different abilities of alkane biosynthesis.
Experimental scheme
We constructed three strains. All the three strains have the same alkane sensing module--AlkSensor with RFP as output. However, they have different abilities of alkane biosynthesis. Strain 1 has the strongest alkane producing ability. The alkane productivity of Strain 2 is less than Strain 1. Strain 3 does not have alkane biosynthesis module.
After systematic analysis of the microbial synthesis pathway from glucose to alkane in E.coli, we decided to choose the pathway from fatty acyl-ACP to alkane as the alkane producing module. The alkane biosynthesis module transferred into E. coli is composed of two enzymes-aldehyde decarbonylase (NPDC) and acyl-ACP reductase (AAR). We conducted codon-optimization to the genes encoding NPDC and AAR based on the work done by Andreas Schirmer, making them more applicable to E.coli. The alkane biosynthesis module is constructed under different constitutive promoter so to generate different ability of alkane biosynthesis.
Figure 1. A pathway of alkane biosynthesis
Strain 1:producing module (constitutive promoter J23114) + AlkSensor
Strain 2: producing module (constitutive promoter J23100)+ AlkSensor
Strain 3:Null+ Alk-Sensor
The promoter strength of strain 1 is stronger than that of strain 2. Theocratically, alkane productivities and intracellular alkane concentrations of the above 3 strains will be different--strain 1 the highest, strain 2 lower and strain 3 the lowest.
Figure 2. Construction scheme of three test strains
The 3 strains are cultured in 3ml M9 medium for about 24 hours. And the result is in accordance with our expectation.
Figure 3. Cell Induced-Fluorescent Indensity
Conclusion:
(1) AlkSensor can respond to the mixed intracellular alkanes, which can be used to detect alkanes inside the cell.
(2) There is a positive correlation between the concentration of intracellular alkanes, and the expression level of the reporter.
