Team:SMS Shenzhen/Contribution
Experiment Report for sacB Promoter Test
1. Experimental Report
The sacB gene usually extracted from Bacillus subtilis encodes the secreted enzyme levansucrase (sucrose: 2,6-b-D-fructan 6-b-D-fructosyltransferase). The enzyme catalyzes hydrolysis of sucrose and synthesis of levans, which are high-molecular-weight fructose polymers. In E. coli, levansucrase activity is mostly located in the periplasm. The molecular basis of the toxicity is still unclear, but the toxicity could be due to an accumulation of levans which might encumber the periplasm because of their high molecular weight or a transfer of fructose residues to inappropriate acceptor molecules, which could thereafter have toxic effects on the bacterial cells. In our project, we make use of the sacB gene to kill the rest of E.coli which didn’t play an effective role in coccid on leaves and scattered into the surroundings.
The sacB gene has been used in iGEM, but we didn’t find much description to sacB gene. Therefore, the efficiency of the promoter of sacB gene, which is a constitutive promoter, is uncleared. This experiment tests the efficiency of the promoter of sacB gene. We designed a plasmid with the promoter of sacB, ribosome binding side J61100, GFP and terminator B0015 on the backbone pSB1c3.
Figure 1 sacB promoter(534bp)+RBS（BBa_J61100）+green fluorescence protein +double terminator（BBa_B0015）+backbone（pSB1c3）
Figure 2 Sequence of sacB promoter
1.2. Vector Construction
We sent the plasmid to genscript company and had it man-made synthesized. To make a measurement next, we also get a standard promoter BBa_J23100 into the identical plasmid backbone.
Figure 3 The standard promoter BBa_J23100 into the identical plasmid backbone
1.3. sacB Promoter Measurement
1.3.1 Abstract
In order to measure the strength of sacB promoter, standard measurement approaches for characterizing the sacB promoter were used so that it will be easier to be re-used by future teams.
We choose BBa_J23100(figure 4) from the Relative Promoter Units (RPUs) as the comparison to the sacB promoter.Three groups were set including an expertimental group(transformed with the sample),a positive control group(transformed with the standard J23100) and a blank controller.In each there were also two parallel tests. After linking up the standard promoter BBa_J23100 into the identical plasmid backbone,we attained the OD600 to estimated the concentration of bacteria suspension with our Linear function of detecting concentration of bacteria suspension in experiment 1. With the data of OD600,we controlled the bacteria in logarithmic phase.Then we made a measurement of the fluorescene of each groups.Using the fluorescein fluorescence standard curve made by A1~12 in 96well plates and three groups of fluorescein intensity data,fluorescenece concentration was computed and histogram of two promoters was made to figure out the intensity difference from fluorescenece concentration.
1.3.2 Method summary and experimental data
1.3.2.1 Measurement of OD600
Using the spectrophotometer to get the estimated value of optical density of the 3groups,we made a conversion to the concentration of the bacterial suspension as an alternative independent variable of the expression intensity with the Linear function of detecting concentration of bacteria suspension in experiment 1Y(Figure 5).Table 1 shows the Bacterial suspension Concentration[(CFU)/g] converted from the optical density[Abs].
Figure 4 Linear function of detecting concentration of bacteria suspension in experiment 1
Table 1
1.3.2.2 Fluorescent Measurement
Controlling the bacteria in logarithmic phase,we prepared the set-up of the ELISA and calibration of fluorescence with each of the triplicates of the 6 constructs, which was measured for its fluorescence. We also designed a set of precise concentration gradient to map the fluorescein fluorescence standard curve, after which the fitting function curve was attained from data in A1~12.
For triplicates in each parallel group, its reading from ELIASA was recorded and the geometric mean of fluorescence [ABS]was calculated to reach error reduction . Table 2 shows the original data from plate reader while table 3 shows the geometric mean of the fluorescence averaged from
Figure 5 Original data from plate reader
Figure 6 geometric mean of fluorescent absorbancy [ABS]
1.3.2.3 Discussion
Considering the linear relation was uncertain, It is hardly to guarantee the correspondence of the GFP expressive quantity and the promoter intensity as what is widely believed. If time allowed, we would make a vertification of the linear relation about the GFP expressive quantity and the promoter intensity in person. Even though the measurement steps were not totally standard as the Interlab, the promoter intensity of sacB met the expectations as its fluorescence concentration surpasses the BBa_J23100 approximately.
Hereto, the expression of the sacB promoter in our engineering E.coli has reach an experimental vertification.
