This part is designed for the acid-tolerant system, consisting of fabB with his tag. FabB: coding sequence for 3-oxoacyl-[acyl-carrier-protein] synthase from Escherichia coli (strain K12). (reference: NCBI locus tag - b2323) This involved in the type II fatty acid elongation cycle and catalyzes the elongation of a wide range of acyl-ACP by the addition of two carbons from malonyl-ACP to an acyl acceptor.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 991
- 1000Illegal SapI.rc site found at 571
Illegal SapI.rc site found at 721
Illegal SapI.rc site found at 1084
Team PuiChing_Macau 2021: Investigation of the acid-tolerant system of our engineered protein
The pH of the environment will have changed due to the presence of lactic acid which can easily inhibit the growth of E.coli, indeed, results obtained the measurement of the food waste shown that many of the food waste from the daily basis state are in pH4-pH7, which reduces the efficiency of PLA production system. Therefore, in order to solve this problem, we designed an acid tolerance system composed of the fabB gene. To investigate the Acid-Tolerant system of our engineered protein, IPTG was added to the bacterial cultures. IPTG addition was achieved at a time of 4 hours to reach the concentration of 1mM and incubate overnight. Briefly, to compare and analyse the growth of e.coli in different pH environments, we conducted overnight culture of a single positive colony from each of our recombinant E.coli strains and get re-transformation to agar plate with different pH environments (pH4 and pH7), and compare it with and without the Acid-Tolerant system (the fabB gene) When comparing the data, colony forming units (CFU) were used to estimate the number of E. coli growing in agar plates. Colony forming units were used to count the microorganisms to be cultured and only live cells were counted. In order to ensure that our samples produce CFU within this range, the samples need to be diluted and plated with several diluents (10-5, 10-6, 10-7 and 10-8).
Total colonies（CFU/mL) = No. of colonies *Total dilution factor/Volume of the culture plate in mL
As shown in Figure 1, we can see that the pH environment does have effects on the growth of the E.coli, there are fewer colonies in pH4 compared to the one in pH7, therefore, it shows that it is necessary for us to add the Acid-Tolerant system. After adding the Acid-Tolerant system, containing with the fabB gene, the total number of e.coli have a significant increase compare to the e.coli without the system in pH4 ( the acidic environment), whereas in pH7, the fabB gene does not have negative impacts on the growth of the E.coli, the growth range is similar in both with and without the Acid-Tolerant system, which proves that the fabB gene does help increase e.coli growth in the acidic environment.
Fig 1. Number of E.coli colonies (WT: BL21 control, FabB: E.coli with acid tolerant gene) in pH 4 and pH 7(N=4, error bar: SEM)
Fig 2. LB agar plate 50μg/mL (E. coli BL21(DE3) in pH 4
Fig 3. LB agar plate 50μg/mL (E. coli BL21(DE3) in pH 7