Team:NTHU Taiwan/Results

Results

Overview

From previous descriptions, we know that lipase can convert lipid into fatty acid. And the lipase activity can change with the temperature. (see the design)

Lipase

Here, we design several experiments to prove the feasibility of our project and analyze some data. The followings are our achievements:

Our vectors successfully constructed

The cold-adapted lipase A is a protein from an Antarctic deep-sea psychrotrophic bacterium Pseudomonas sp. 7323. Lipases are glycerol ester hydrolases that can hydrolyze ester to free fatty acid and glycerol. With overexpression of LipA, the bacteria can produce different concentrations of fatty acid under different temperatures.

We get the sequence of Lip A from NCBI. To check the expression of Lip A in cells and facilitate the purification of this protein, we attached the 6 X His tag on the C-terminal of this protein. This part was inserted into the iGEM provided expression vector psB1C3 through the restriction site EcoRI and SpeI (Fig1).

Figure 1. Construction of expression vector pSB1C3-LipA-6X His-tag. The insert sequence is flanked by EcoRI and SpeI restriction site.

This recombinant plasmid was further screened by ampicillin selection, colony PCR in the cloning E. coli, DH5α (Fig. 2) and the digestion of miniprep product (Fig 3). From those results, we can prove that the Lip A sequence synthesized by IDT was successfully integrated into the cloning vector psB1C3.

Figure 2. DNA electrophoresis with 1.32% gel was performed to screen the positive recombinant. The plasmid constructed was 4176bp and the predicted PCR result should be 2420bp (flanked by the VF2 and VR primer). Lane 1: DNA loading marker, Lane 2-7: VF2 and VR PCR product).

Figure 3. DNA electrophoresis with 1.32% gel was performed to screen the positive recombinant. The plasmid extracted was digested with EcoRI and SpeI. The digested part (R0010-Lip A-6X His tag) should be 2129bp. Lane 1: DNA loading marker, Lane 2 to 5: Plasmid digested, Lane 6: J04450 control. The plasmid should be digested into 2 parts, one is R0010-Lip A-6X His tag, the other one is the psB1C3 backbone, both parts are about 2200bp. Thus, there will be only one band on the lane.

Lipase successfully produced

We have transformed E. coli BL21 strain with pSB1C3-LipA-His tag construct which has been previously proved successful. The positive transformants were screened with ampicillin and colony PCR. We liquid cultured the cells and collected after 16 hours. After washing with PBS, we lysed the cells by using lysis buffer (please refer to the Experiment protocol) to get the protein lysate. Western Blot has been performed to check the expression of Lip A by using an antibody against His tag.

Figure 4. Western Blot analysis of total protein extracted from BL21 transformed with pSB1C3-Lip A-6X His tag by using antibody against His tag. Lane 1: BL21 control with no plasmid transform. Lane 2 and 3: Protein loading marker. Lane 4 to 13: BL21 transformed with our construct. According to the information on UniProt, Lip A from Pseudomonas sp.7323 was reported to be 64.555 kDa.

Our enzymes are functional

In order to verify the functionality of Lip A expressed, we have done a functional assay with the protein extract from BL21 by sonication. To determine the lipase activity, we utilized a spectrometry-based method by using 4-nitrophenyl decanoate as a substrate. The amount of 4-nitrophenol hydrolyzed and released was determined spectrophotometrically at 405nm. We compared the fluorescence and found that the change in fluorescence is greater in the protein in BL21 expressed Lip A compared to control (Fig. 5).

Figure 5. Lipase activity assay was performed to analyze the function of lipase A. The protein was first incubated at 30°c for 30 minutes at pH9.0. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm 30°c in continuous duration (0, 20, 40, 60 mins). The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

Enzyme activity matches our expectation over temperature change

Since our goal is to build a precise thermal-tunable promoter with a dynamic range of gene transcription, the lipase activity in a variety of temperature is very important to us. Hence, we evaluated the lipase activity at different temperature and the curve illustrated fitted to what reported in previous research of Lip A of Pseudomonas sp.7323 (Figure 6 and 7).

Figure 6. Lipase activity assay analysis was performed to check the activity of lipase at different temperature varies with time at pH9.0. The protein was first incubated at the experiment temperature (10, 20, 25, 30 and 40°c) for 30 minutes. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm in continuous duration (0, 20, 40, 60 mins) at the temperature required. The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

Figure 7. Lipase activity assay analysis was performed to check the activity of lipase at different temperature for 40 minutes at pH9.0. 40 minutes of data were chosen because this time duration is best fitted to the activity curve reported previously in the paper. Thus, our lipase is proved to be functional.

Fatty acid sensitive promoter

We tried to improve the original fatty acid promoter, pFadBA, by increasing the strength and decreasing the leakage of the promoters. We tested several characteristics of these promoters:

1. Effect of fatty acid concentration on promoter
2. Decrease in leakage compare to pFadBA
3. Strength enhance compare to pFadBA

To compare our results, we divided our promoters into five groups based on their structure and design concept: (for detailed design of promoters, check parts)

Promoter pFadBA modification on consensus sequence

Though both pFadBA_NTHU (BBa_K3040005) and pFadBA_NTHU (BBa_K3040006) mutant have 1 to 2 fold increase in expression over native pFadBA as the concentration of fatty acid rises, pFadBA_NTHU has greater performance than pFadBA_NTHU mutant.

Thus, we could conclude that the modification of consensus sequence in -10 and -35 region could indeed improve the expression of the native promoter pFadBA, while the point mutation in advance had probably negative effects on its performance.

Figure 8. Relative protein expression of fatty acid promoter pfadBA-NTHU and pfadBA-NTHU mutant after 4 hours of induction under different fatty acid concentrations (n=3).

Hybrid pFadBA promoter with Lac and additional FadR binding sites

As we can see from fig.2, the expression of PFL1 (BBa_K3040011) was greatly enhanced to 2 folds under IPTG induction. Yet, there is no difference between the inductions of IPTG in PFL2 (BBa_K3040012). Therefore, PFL1 is proved to successfully express while PFL2 fails to function.

Here, we conjecture that two fadR binding sites might have been over repressing the expression of the promoter. Higher fatty acid induction might help, yet, the solubility of oil has limited us for further experiments.

Figure 9. IPTG induction effects on protein expression of fatty acid pFadBA-Lac-2 (PFL2) after 16 hours of induction under 5mM fatty acid (n=3).

Figure 10. Relative protein expression of fatty acid pFadBA-Lac-1 after 16 hours of induction under different fatty acid concentrations (n=3).

Replacement of pFadBA promoter with rrnD promoter, UP element, and additional FadR binding sites

The result turned out to be frustrated that none of them function. We had proposed some reasons that might lead to failure. UP element might have caused some conformational change of the promoter, there might be too many changes at the same time, or the fadR binding site might not function well with the rrnD promoter. Yet, we can’t figure out what has happened inside the cell. (Fad BS1-BBa_K3040008, Fad BS2-BBa_K3040009, Fad BS3-BBa_K3040010)

Figure 11. Protein expression of fatty acid promoter TP24-rrnD-fadRBS1, TP24-rrnD-fadRBS2, TP24-rrnD-fadRBS3 after 16 hours of induction under 5mM fatty acid (n=3)

Replacement of pFadBA promoter with pFadD promoter, Lac, and additional FadR binding sites

We can see clearly that pFadD_Lac (BBa_K3040014), pFadD (BBa_K3040013) promoter with an additional lac binding site, has relatively low leakage and has 2-3 fold increase in expression as the fatty acid concentration rises. Though compared to the original promoter pFadBA, both pFadD and pFadD_FadR (BBa_K3040015) also has reduction in leakage, we assumed them not functioning since they show no changes in expression as the concentration of fatty acid rise.

Figure 12. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under 5mM fatty acid (n=3).

Figure 13. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under different fatty acid concentration (n=3).

Combination of gene of TesA protein with pFadBA promoter

The fold change of fluorescence after 16 hours 5mM oleic acid induction can come up to 9-fold. This has greatly improved the native strength of the promoter since it can only increase to about 2-fold. This modification helped us to control the strength of promoter more precisely compared to the native pfadBA since the induced-transcription range of the promoter has been broaden. (TesA_FadBA is BBa_K3040007)

Figure 14. Protein expression of fatty acid promoter TesA-FadBA after 16 hours of induction under different fatty acid concentration (n=3).

We have successfully proposed five new functional biobricks (BBa_K3040005, BBa_K3040006, BBa_K3040007, BBa_K3040011, BBa_K3040014) with improvements in the original pFadBA promoter in leakage reduction and strength enhancement.

Conclusion

We demonstrated that our cold-adapted lipase and fatty acid promoter could be used to construct the Tunable-Temperature-Sensitive-System (TTSS). We proved that the catalytic activity changes over temperature and that the new promoters we designed had better expression.

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