Team:Thessaly/Contribution

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CHARACTERIZATION.
Aim Constructs' creation Method Results

This year, iGEM didn’t 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 beta-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 beta-lactamase, we wanted to determine the expression levels of this enzyme under the control of different promoters from the Anderson family 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 turns from yellow (380nm) to red (490nm) when hydrolyzed by b- lactamase. We then take quantitative results though frequent plate reader measurements.

Constructs' creation

For our experiments we chose four promoters from the Anderson family with different expression strengths, which were always combined with our characterization part, the beta-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 beta-lactamase coding sequence and a terminator, was ordered from IDT as one. Subsequently, the different promoters and biobrick prefix were incorporated by PCR as overhangs to primers annealing to the ordered part. After the successful PCR (Figure 2), ligation to pSB1C3 and transformation into E. coli DH5a cells, BL21 (DE3) cells were transformed with the desired structures 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 overhang primers 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 valuable and reproducible data, we performed 2 biological and 3 technical replicates for each construct. To ensure that the absorbance measured corresponds only to the enzymatic activity of beta-lactamase, 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 the third 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 (did triplicates) 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.

We took absorbance measurements every 30 secs for 2 hours at 490nm for the hydrolyzed nitrocefin substrate, and at 600nm for the cell growth. Since the results do not change after the first fourth 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 b-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, 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 beta-lactamase 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 from previous teams that measured fluorescence and the same pattern is observed. The controls conditions (pSB1C3 and BL21, or light purple and light blue respectively) confirm that the absorbance measured derives from beta-lactamase activity only.

Figure 4. The observed color change due to the hydrolyzation of nitrocefin due to the production of b-lactamase.