What we have accomplished
1. Successfully verified existing part BBa_K325909, luxCDABEG (abbreviated as 4L), and confirmed that it works.
2. Established an optimal condition of Arabinose concentration to induce lux-operaon expression
3. Study the function of luxG and construct 2 new parts, one basic one-luxG and one composite one luxCDABEG-luxG (abbreviated as 2G), and found luxG cold improve the bio-luminescence strength of lux operon in bacteria
4. Constructed luxCDABEG and luxCDABEG-luxG into pHB vectors and confirmed through electrophoresis
5. Transformed plasmids to agrobacteria (pHB-luxCDBAEG and pHB-luxCDABEG-luxG)
6. Conducted agrobacterium mediated transformation and injected target plasmids 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.
Establish a method to compare bioluminescence strength in the bacteria
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.
luxG could improve the bio-luminescence strength of lux operon in bacteria
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
Transforming target plasmids to agrobacteria
We succesfully transformed target plasmids got in step 4 into agrobacteria and verified by colony PCR and then positive colonies were cultured for further study. We designed a pair primers of luxB for colony PCR and pick single colonies (showed as below pic) as template to amplify for verification.
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.
After the injection, the plants showed no bioluminescent abilities. We have done this for three times, but haven’t observed any light of the plants. Plant is very complicated to conduct gene modification. We’re not very sure about the reason. Since we have done every previous step successfully, this result may be explained by the non-uniformity of plants.
1. Continue injecting plants with 2G
2. Research other conditions that can lead to a greater bio-luminescence and design the solution