Team:Thessaly/Characterization
This year, iGEM did not hold an InterLab study but instead introduced a new measurement criterion; the addition of quantitative experimental characterization data to an existing Part from the Registry of Standard Biological Parts. In our effort to provide new experimental data related to our project, we chose to characterize the expression of the β-lactamase gene by using different promoters derived from the Anderson promoter family. In this page, the whole experimental process that we followed for the contribution is displayed, together with specific links to the Registry of Standard Biological Parts.
Aim
As our test’s reporter gene is β-lactamase, we wanted to determine the expression levels of this enzyme under the control of different promoters from the Anderson family of constitutive promoters, in order to have a clear image about our enzyme’s activity. To do so, we performed an enzymatic assay in an in vivo expression system. We used the chromogenic substrate nitrocefin, which changes color from yellow (380nm) to red (490nm) when hydrolyzed by the β-lactamase enzyme. We then gathered quantitative results for the hydrolysis of our substrate, Nitrocefin, though frequent microplate reader assays, after the in vivo expression of the β-lactamase enzyme regulated by the abovementioned promoters.
Constructs' engineering
For our experiments we chose four promoters from the Anderson family with different activities, which were always combined with our characterization part, the β-lactamase (BBa_I757010) coding sequence, and a common terminator, resulting to four different constructs (test devices) as indicated in Table 1.
Table 1. The Registry Parts used for the generation of our newly designed BioBricks for our characterization experiments.
The common part of the four devices, which contains the RBS, the β-lactamase coding sequence, the terminator and the suffix, was ordered from IDT as one. Subsequently, the different promoters and BioBrick prefix were incorporated by PCR as overhanging sequences to primers annealing to the ordered part. After the successful PCR reaction and incorporation of the promoters' sequences(Figure 2), ligation to pSB1C3 vector and transformation into E. coli DH5a cells, BL21 (DE3) cells were transformed with the vectors containing the different constructs for optimal protein expression.
Figure 1. The designed primers containing the sequence of the different promoters for our overhang PCR
Figure 2. The results obtained after the PCR with the primers containing the overhanging sequences for the different promoters of the Anderson family. We tested different annealing temperatures (45, 47 & 53℃) aiming for clear results. The expected band is at 1119bp and the ladder used was the 100bp DNA ladder by NEB.
Method
In order to produce measurable and reproducible data, we used 2 biological and 2 technical replicates for each construct. To ensure that the measured absorbance corresponds only to the enzymatic activity of β-lactamase enzyme, we included 3 controls in our experiments. The first control contained M9 medium only (no cells) and nitrocefin, the second empty BL21 (DE3) cells (no plasmid) and nitrocefin, while in the third control, BL21 (DE3) cells contained the plasmid, but not the part (no insert). The workflow was performed as described below:
1. We grew 7 cultures (four different cultures containing one of the four devices each time and three controls) overnight in 5ml LB (~16h) at a shaking incubator, 37℃ / 210rpm
2. The following morning, we measured the OD600 of the overnight cultures
3. We diluted all cultures to OD600 = 0.05 in M9 medium
4. We then grew cells at 37℃ /210 RPM until OD600=0.4-0.6 (~2h)
5. We diluted all cells to the same OD600 (e.g. 0.4)
6. We loaded 180ul of culture in a 96-well plate (2 technical results) and 20ul of the nitrocefin substrate, in order to perform the assay.
7. We finally measured the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) in a microplate reader. We shook between measurements.
The absorbance measurements were conducted every 30 secs for 2 hours at 490nm for the measurment of the hydrolyzed substrate (Nitrocefin), and at 600nm for the cell growth. Since the results do not change after the first quarter of the assay due to substrate limitation, the first 25 minutes are depicted.
Results
Figure 3. The hydrolysis of nitrocefin enabled by the expression of the β-lactamase gene, under the control of different promoters (J23100, J23105, J23106 & J23119) of the Anderson family. The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.
The graph above shows the substrate (nitrocefin) hydrolysis (490nm) divided by cell growth (600nm), to normalize all values. Since all conditions except the promoters remain the same, differences in the curves indicate different expression levels attributed to the promoters' activities, respectively. Even if the experiments were conducted for 2 hours, after the first 5 minutes the reaction is complete and plateau is reached. The maximum expression of the β-lactamase enzyme was observed under control of the J23119 (brown line) which is the wild type promoter of the Anderson family. The expression is reduced with the J23100 and J23106 (yellow and purple line respectively), while the lowest expression levels were observed with the J23105 promoter (blue line). These results are in accordance with those of previous teams that measured fluorescence (the same pattern was observed). The control conditions (pSB1C3 and BL21, or light purple and light blue respectively) confirm that the absorbance measured derives from the activity of the β-lactamase enzyme only.
Figure 4. The observed color change due to the hydrolysis of nitrocefin due to the production of the β-lactamase enzyme.
