Team:ShanghaiTech China/LightControl
1. The principle of pDawn
The pDawn system is a robust gene expression system acting as a light-activated manner. Namely, blue light can activate the expression of target gene, which can be greatly enhanced with either the increase of light intensity or the extension of the duration of illumination. The principle of the system at a micro-level is elucidated as below.
The system features the histidine kinase YF1, which employs a light-oxygen-voltage blue-light photosensor domain. That is, in the absence of the blue light, YF1 phosphorylates its cognate response regulator FixJ, which then drives significant gene expression from the FixK2 promoter and upon light absorption, the expression can be therefore suppressed. In the system, all components of the YF1/FixJ TCS were assembled on the single medium copy plasmid pDawn in which YF1 and FixJ are constitutively expressed from the LacIq promoter as a bicistronic operon. Then, target genes can be introduced in a single step via a multiple-cloning site (MCS) downstream of the pFixK2 promoter. After that, the insertion of the λ phage repressor cI driven by Fixk2 represses λ promoter pR in pDawn, inverting signal polarity and renders gene expression light activated. That is, with the appearance of blue light, FixK2 is suppressed so that cI will also be suppressed. In this case, the expression of the target gene can be initiated by the λ promoter pR.
2. The light-inducible NAS plasmid: pDawn-NAS
We intended to use light to induce NAS expression instead of IPTG so that we can easily program light to control the production of N-palmitoyl serinol. To replace the lac operon with a light-inducible promoter, we introduced the blue-light inducible system called pDawn into our system and constructed the pDawn-NAS fusion plasmid.
For cloning, we used PCR with a high-fidelity enzyme to get the linear pDawn vector (7198 bp) and NAS gene fragment (1685bp) with overlapping sequence. As shown above, the PCR product was separated by agarose gel electrophoresis, then gel extraction kit was used to isolate the target DNA fragments. After that, the Gibson Assembly Cloning kit was used to recombine the vector and fragment into the final plasmid pDawn-NAS. The recombination product was then transformed into DH5α competent cells. Finally, we got our desired pDawn-NAS plasmid. The sequence map was shown below. The recombinant plasmid was then transformed into BL21:DE3 bacteria for gene expression.
The production of N-palmitoyl serinol by pDawn-NAS
As we have got our desired plasmid, we wanted to test whether NAS gene can be expressed to produce N-palmitoyl serinol in the pDawn system. Therefore, we directly used the LC-MS to determine the production of N-palmitoyl serinol. We inoculated 100 ul overnight cultured bacteria fluid into 10 ml LB with kanamycin (50 ug/ml) and cultured at 37 °C with shaking (220rpm) for 3 h until OD550 reaches 0.6-1.0. Then we poured all the LB into a petri dish and exposed the petri dish with a blue light at 29 °C for 24 h. Next, we centrifuged down 1 ml bacteria to get the supernatant and added equal volume ethyl acetate to it. The ethyl acetate solution was finally used for the LC-MS. As shown bleow, the introduction of NAS in the pDawn successfully induced a strong signal around t = 4.12 min. Yet, the empty plasmid control showed little signal there.
We confirmed the t = 4.12 min peak is the N-palmitoyl serinol as the following.
We fragmented the N-palmitoyl serinol by giving a certain amount of energy, and then detected the Mass/Charge value of each fragment. The results of standard N-palmitoyl serinol and the sample of small molecules at t = 4.12 min were shown blow. They had nearly the same characteristic patterns in peak positions and the ratio of intensity, which proved that the signal at t = 4.12 min was generated by N-palmitoyl serinol. Thus, we successfully confirmed that the production of the desired molecule via the pDawn-NAS.
3. Characterization of pDawn system
As the mass spectra only provided a measure of the relative amount of N-palmitoyl serinol, we decided to use mEGFP to precisely measure the properties of the pDawn system under different light inducing conditions. Therefore, we constructed pDawn-mEGFP plasmid with mEGFP fluorescence Intensity (F.I./a.u.) as the quantitative readout.
Results of the characterization
In this part, 488-nm blue light was applied to induce mEGFP expression and the light intensity was 9.6uW. To examine the dynamics of mEGFP expression, different illuminating durations (6h, 12h, 24h, 30h, 36h) were used in our experiment. Unless stated otherwise, all experiments were carried out with E. coli BL21:DE3 pLysS strain in M63 or SOC medium containing 50 μg/ml kanamycin. The detailed protocol was listed at the "PROTOCOL".
Results were analyzed in both visualized micrographs and normalized line graphs, which showed the qualitative and quantitative mEGFP expression level respectively.
Micrographs are listed as follows:
As the illumination duration increases, the bacteria densities also increased until it became stable after 30 hours. Substantially, there were also big increases in mEGFP fluorescence per bacterium in light-on groups over light-off groups, suggesting the light induction was successful.
The graph shows that mEGFP expression was sharply increased after 6 hours when treated with light, and reached the maximum expression level after 24 hours. Although mEGFP expression in the light-on groups were much higher than that in light-off groups, there was significant leakiness expression in darkness. To overcome this deficiency, several modifications were applied to the plasmid, which will be elucidated in the next part.
4. Characterization of Modified pDawn system
As mentioned above, we have obtained five newly modified pDawn-mEGFP plasmids, with four modified with promoters with potentially higher efficiency (mtac, T7c, tac, 100) and one displaced its histidine kinase YF1 with YF2 ( YF2).
In this part, we compared the performance of the new constructs with the original pDawn-mEGFP component based on both the efficiency of mEGFP expression and the leakage level. To examine mEGFP expression dynamics, different illuminating duration was used in our experiment.
Results of the characterization
Micrographs are listed as follows:
Comparing with the original pDawn-mEGFP, both mtac and YF2 featured an ideal expression capacity and a minimum leakage level. As for T7c, the absolute fluorescence intensity reached a relatively high level in a short time, namely reacting faster to light induction, even though the leakage level was not reduced. As for the other 2 modified bacteria, they were not idealistic because they introduced more leakage while lowering the overall fluorescence intensity.
In conclusion, T7c, mtac and YF2 were more suitable candidates in certain aspects of our project. Thus, they were further analyzed with tac as the negative control and pDawn-mEGFP as the original control.
Firstly, we quantified the absolute expression level of the T7c.
A significant increase of the absolute expression level compared to the original pDawn-mEGFP can be observed directly from the graph in the first several timepoints, indicating T7c's good property of rapid reaction with time.
After that, we compared YF2, mtac with pDawn-mEGFP as below.
Comparing with the original pDawn-mEGFP, a similar effect can be observed in YF2 and mtac. To quantify the expression efficiency and leakage level, further analysis was carried out as below.
The effect on reducing leakage level was proven with the following graphs.
Figures above clearly show the decreased background expression level of mtac and YF2 compared with pDawn-mEGFP. Specifically, the YF2 maintained a relatively low level of leakage which also dipped over time, while mtac showed a downward trend after 12h before becoming stable after 30h. The lowest leakage of the mtac and YF2 occurred at 12h and 36h respectively, with mtac reducing the leakage by 78.74% and YF2 by 78.98% when compared with pDawn-mEGFP.
Expression efficiency was compared in the following graphs:
Comparing with the original pDawn-mEGFP, a similar expression efficiency can be observed in YF2 and mtac and the timepoint of 12h and 36h witnessed a substantial climb of the YF2 and mtac respectively, exceeding pDawn-mEGFP by approximately 50%.
As a result, mtac and YF2 can be characterized as the most ideal modified components among all of the candidates, with similar efficiency as the original one and better performance on leakage reducing. Though the expression level of the 2 modified components is not yet ideal enough, we can expect a potential more excellent result obtained under suitably higher light intensity or a more proper inducing environment, which will be our plan for the future.
T7c highlighted a significant increase of the absolute expression level compared to the original pDawn-mEGFP, reacting rapidly with a short time range. As for the leakage problem, we expect a combination of T7c with other low leakage modified components such as YF2 to weaken the leakage while obtaining a relatively high expression level.