Overview

What we have accomplished

1. Successful synthesis of parts BBa_K325909, luxCDABEG (abbreviated as 4L), verification of the part and confirmation that it works.

2. Establish an optimal condition of Arabinose concentration

3. Study the function of luxG and construct a new part, luxCDABEG-luxG (abbreviated as 2G)

4. Construct luxCDABEG and luxCDABEG-luxG into pHB vectors and confirm through electrophoresis

5. Transformation plasmids (pHB-luxCDBAEG and pHB-luxCDABEG-luxG)

6. Agrobacterium mediated transformation and injection of this into Nicotiana tabacum(tobacco plant)

What we have not accomplished

1. Detect any light in the plants injected with pHB-luxCDBAEG or pHB-luxCDABEG-luxG

Verification of luxCDABEG (4L)

We extracted the plasmid from the kit plate and conducted transformation. Through conducting a gradient test with three trials, we have confirmed that luxCDABEG works.

Figure 1: Raw Data Recording Luminescence and Abs at Different Times

Figure 2: Processed Data Recording Relative Luminescence of 4L at Different Times

From the graph, an S trend can be observed, where there is a slight increase in luminescence in the first 2.5 hours with a sharp increase right after, and eventually the rate of increase of luminescence decreases. By modeling the graph, a clearer pattern can be derived and a prediction of later hours can be made, with the luminescence reaching a plateu (refer to model page for a detailed explanation).

Other than conducting a gradient test for luminescence, we also put Arabinose in a cylindrical flask of luxCDABEG to test if it works. The glow of the bacteria confirms the ability of the gene to induce bioluminescence.

Establishment of Optimal Arabinose Concentration

Conducting a gradient test once again, we aimed to find the optimal Aribinose concentration where there is greatest bioluminescence. We used five different concentrations of Arabinose (0M, 0.1M, 1.1M, 1.5M, and 2 M) and plotted their luminescence with time.

Figure 3: Raw Data Recording Different Arabinose Concentration and Luminescence with Time

Figure 4: Processed Data Recording Relative Luminescence of Different Arabinose Concentration With Time

The graph shows that the optimal Arabinose concentration that would lead to the greatest bioluminescence is 0.1 M. The graph also shows that other concentrations of Arabinose yield very similar luminescence, which shows that there is a very specific range of optimal Arabinose concentration.

Study the Function of luxG and Construct a New Part

We have modified the part BBa_K325909, luxCDABEG (4L), so as to create a new part, luxCDABEG-luxG (2G). By comparing the luminescence of the two, we were able to determine the function of luxG and to create a new sequence that allows for a greater bioluminescence. This comparison is also measured through a gradient test.

Figure 5: Raw Data Recording Luminescence for 2G and 4L in Different Times

Figure 6: Processed Data Recording Relative Luminescence for 2G and 4L at Different Times

The graph shows that the new sequence our team modified, luxCDABEG-luxG (2G), allows for a greater bioluminescence. Because the only difference between the 2G and 4L is the luxG, it can be reasonably concluded that luxG is the reason behind the increase in bioluminescence (refer to improve page for a detailed explanation).

We also added Arabinose to a cylindrical flask with 2G, so as to confirm our method is working and that the luminescence is greater.

Construction of pHB-luxCDABEG and pHB-luxCDABEG-luxG

After inserting luxCDABEG and luxCDABEG-luxG into pHB vectors, we used gel electrophoresis for verification.

Figure 7: pHB-luxCDABEG

Figure 8: pHB-luxCDABEG-luxG

Transformation plasmids (pHB-luxCDBAEG and pHB-luxCDABEG-luxG)

Agrobacterium mediated transformation and injection of Nicotiana tabacum

The last step in the process includes the agrobacterium mediated transformation as well as the injection of the sequence into Nicotiana tabacum.

Future Plans

1. Continue injecting plants with 2G

2. Research other conditions that can lead to a greater bioluminescence