Difference between revisions of "Team:NYMU-Taipei/Nitrogen starvation"

　　With the development of global economy in the latest demands of energy in world, an average Taiwanese produces around 2.58 metric tons of carbon emission a year. This number far surpasses those of China, Japan and South Korea. Our project, as a result, works to use biofuel as an alternative fossil fuel to reduce the current energy crisis. In our lab, we use microalgae as the source of biofuel since they have the greatest capability of producing large amount of oil.

　　Our project aims to increase oil accumulation through nitrogen starvation, which was brought up in a research we came upon. Under nitrogen starvation, de novo synthesis of triacylglycerol from acyl-CoA increases. Acyl moieties derived from the degradation of membrane lipids then recycle into triacylglycerol, increasing carbon flux towards glycerol-3 -phosphate and acyl-CoA for fatty acid synthesis. As such, the oil accumulation under nitrogen starvation will increase.

　　There are currently two types of cultivated systems: open-pond and closed bioreactors. While open-pond costs way lower than closed bioreactors, open-pond cultivate microalgae is with significantly lower oil contents. Many microalgae farms today cultivate microalgae in closed ponds, where regulations are made to keep the nitrogen level low. This method, though, consumes lots of energy due to the need to maintain proper temperature, nutrition, light, and other growing factors. We want to develop a new method that allows microalgae to reach nitrogen starvation in open-pond, and thus reaching the same effectiveness of closed bioreactors with the affordable price of open-pond.

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Design

　　NrtA protein sticks to the periplasmic membrane through a flexible linker to capture nitrite or nitrate in the periplasm. Then delivery to the transmembrane complex that made by NrtB. In our project, we try to transform NrtA gene from cyanobacteria Synechocystis sp. PCC 6803 to E.coli, and then co-culture the engineered E.coli with microalgae. Engineered E.coli will be capable of clutching nitrite or nitrate present in the environment. They will not intake nitrate or nitrite since the gas accumulation may be lethal to cells. But the amount of cells that contain nitrite will decrease. Therefore, the microalgae will undergo nitrogen starvation and produce oil more efficiently.

　　After building up the nitrogen starvation and extracting oil from microalgae, we need to kill E.coli to prevent contamination. So we plan to use endolysin and holin for cell lysis, which is similar to the mechanism used by team PeKing (2014 iGEM Beijing). Holin can trigger the formation of holes on cell membrane. When holin successfully forms holes on cell membrane, endolysin can pass through the membrane to decompose peptidoglycan. E.coli is lysed after the cell membrane and cell wall are destroyed. To control the suicide timing, we designed an inducible promoter for holin, so that we can induce E.coli suicide at the exact time we want.

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Experiments

NrtA Expression

　　We successfully transformed NrtA gene from cyanobacteria Synechocystis sp. PCC 6803 to E.coli.

　　This figure is restriction enzyme check electrophoresis result of NrtA construct. We use XhoI and HindIII to digest the plasmid, and the expected length is 892bp, 1169bp, 1540bp. M represents 1kb marker, and the result shows that number 2, 4, 6, 8, 10, 12 are right.

Endolysin Construct

　　We successfully constructed J23106-B0034-Endolysin-B0010-B0012. The endolysin in this part is from iGEM released part, BBa_K112806, which is endolysin from enterobacteria phage T4. Besides endolysin, in this composite part, we choose BBa_J23106 as a constitutive promoter, BBa_B0034 as ribosome binding site, BBa_B0010 and BBa_B0012 as double terminator, all of which are widely used parts in iGEM.

This figure is electrophoresis result of endolysin PCR product. The marker is 100bp. The length is 514bp as expected.

This figure is electrophoresis result of endolysin backbone PCR product. The marker is 100bp. The length is 2268bp as expected.

Holin Construct

　　We successfully constructed R0010-B0034-Holin-B0010-B0012. To control the precise suicide timing, we choose a lactose-induced promoter, BBa_R0010, to regulate this suicide mechanism. Besides holin and inducible promoter, in this composite part, we also choose BBa_B0034 as ribosome binding site, and BBa_B0010 and BBa_B0012 as double terminator.

This figure is electrophoresis result of holin PCR product. The marker is 100bp. The length is 657bp as expected.

This figure is electrophoresis result of holin backbone (from BBa_J04450) PCR product. The marker is 1kb. The length is 2458bp as expected.

Endolysin-Holin Construct

　　We successfully constructed R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012. We combined holin and endolysin for suicide mechanism. In this composite part, holin functions as an important regulation role.

This is restriction enzyme check electrophoresis result of holin-endolysin construct. The marker is 1kb. We use XmaI to digest the sample. The total length is 3843bp as expected.

Endolysin-Holin-NrtA Construct

　　We successfully constructed R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012-J23118-B0034-NrtA-B0015. This part combined holin, endolysin, and NrtA and had nitrate-capturing function and suicide function.

This figure is restriction enzyme check electrophoresis result of endolysin-holin-NrtA construct. We use XmaI to digest sample 1~3. The result shows that 2 and 3 are right. M represents 1 kb marker.

*See our parts: click

*See our experiments protocols: click

Growth Curve Measurement

　　In order to investigate the optimal time to co-culture our modified E.coli and Chlorella vulgaris, evaluate growth of Chlorella vulgaris before Nile Red staining for oil determination in microalgal cells, we will measure the OD value at 680nm by using the spectrophotometer and analyze the growth curve in R.
　　However, we encountered some difficulties such as irregular measurement time and personal errors. Thus, we decided to search for better measurement method. Later, we borrowed a photo-bioreactor from Professor Ya Tang Yang, Department of Electrical Engineering, National Tsing Hua University, and used it to measure OD value for more precise bacterial growth curves.

The above line chart presents growth curve of Chlorella vulgaris. This measurement lasted more than 216 hours and it roughly meets our expectation.
Although the later part of growth curve shows violent fluctuating range, it may be affected by environmental nitrogen metabolites from Chlorella vulgaris.
The measurement result helps us determine lipid production of Chlorella vulgaris, and the time we add NrtA-transformed E. coli into the medium to establish a co-culture system.

Automatic Measurement

　　In the photo-bioreactor, there are four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth expression without human intervention. We measure two kinds of microalgae during cell growth in same culture medium, BG-11.

　　The photo-bioreactor was designed by Professor Yang. It was made with Arduino and some circuit components. Yang’s students also helped us assemble and teach us how to use this device. The photo-bioreactor itself can detect multiple units of organisms at same time, has pumps, fans, stir bars and some light bars. In Yang’s laboratory, we created a calibration curve for correcting and confirmed that there was a very high degree of correlation between voltage and OD value. This can be observed in the charts below.

And here are our results.

The above line chart presents growth curve of Chlorella vulgaris, under measurement near 10000 minutes made by the photo-bioreactor.
In comparison to the stimulation model we made, the result meets our expectation.

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NrtA Functional Test

　　We expect the engineered E.coli with NrtA gene could express NrtA protein. When we co-culture it with microalgae, the NrtA protein will capture nitrate and nitrite, and the microalgae will undergo nitrogen starvation and produce more oil. To test our hypothesis, first, we should verify NrtA protein could capture nitrate and nitrite, and then second, test whether the engineered E.coli could decrease nitrate and nitrite in the environment.

NrtA Protein

　　To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to isolate NrtA protein, and used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium with NrtA protein. (You can see the detail in our Notebook page: click.)

　　Here are our results.

Figure1. Nitrate concentration of cell lysate.
Blank: nitrate concentration assay kit assay buffer.
BG11: microalgae culture medium buffer.
CC: competent cell.
NrtA: isolated NrtA protein.

Table1. Dunnett’s T3 test of nitrate concentration of cell lysate.
Blank: nitrate concentration assay kit assay buffer.
BG11: microalgae culture medium buffer.
CC: competent cell.
NrtA: isolated NrtA protein.

　　The nitrate and nitrite concentration of competent cell and NrtA is significant different. The results indicate that NrtA protein can capture nitrite and nitrate! This is a milestone of our project!

The Engineered E.coli with NrtA Gene

　　Then we wanted to know whether the engineered E.coli could decrease nitrate and nitrite in the environment. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered E.coli liquid culture.

Here are our results.

Figure 2. Nitrate concentration of cell.
Blank: nitrate concentration assay kit assay buffer.
BG11: microalgae culture medium buffer.
CC: competent cell.
NrtA: the supernatant of the engineered E.coli liquid culture.

Table 2. Dunnett’s T3 test of nitrate concentration of cell.
Blank: nitrate concentration assay kit assay buffer.
BG11: microalgae culture medium buffer.
CC: competent cell.
NrtA: the supernatant of the engineered E.coli liquid culture.

　　The nitrate and nitrite concentration of NrtA and competent cell has slight but not significant difference. The results imply that NrtA protein could capture nitrate and nitrite while the engineered E.coli with NrtA gene cannot. The engineered E.coli with NrtA gene can express NrtA protein, while NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell does not change a lot. To make NrtA protein become a secreted protein, we are trying to construct signal peptide sequence, so that NrtA protein can be released to the medium.

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Suicide Mechanism Functional Test

　　Before we construct our suicide mechanism with NrtA, we want to make sure that our suicide mechanism, endolysin and holin, can be induced by lactose efficiently. Therefore, we test our suicide mechanism by adding different concentration of lactose in order to find out the minimum effective concentration. Furthermore, to induce E.coli suicide at the time we want, we also want to know the changing degree of the OD value of bacterium at different time after the lactose is added.

　　We measure the OD₆₀₀ value of the sample, and then aliquot liquid culture into several cuvettes. Next, we add different amount of 0.5mM lactose into cuvettes to form different concentration of lactose, and measure the OD₆₀₀ value per hour.

　　As the figure shows, the suicide mechanism is induced immediately when lactose is added into the samples. Besides, we can see that the lactose concentration and the degree of decline of OD value are positively correlated.

　　We also collaborate with team TAS Taipei. They help us by testing our holin-endolysin-NrtA construct.

　　As the figure shows, the trend of the relative absorbance is downward as the lactose is added to induce the suicide mechanism. The concentration of lactose is also positively correlated with the declining degree of relative absorbance.

　　We need to mention that there are some differences in the process of team TAS Taipei’s and ours functional tests. We use different medium to culture the bacterium. For the bacteria with holin-endolysin construct, we use Luria-Bertani (LB) broth with 0.9% glucose to culture; team TAS Taipei cultures the bacterium with holin-endolysin-NrtA construct with only Luria-Bertani (LB) broth. According to our previous experiment result, the bacteria don’t grow well in only LB broth medium. Therefore, to make the figure look similar, team TAS Taipei normalizes their data (absorbance at 0 hour is the background). Despite the different of culturing medium, the results both show that our suicide mechanism can work. (see more detail about collabration)

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Reference

J.W. Allen et al. (2015). Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae Coccomyxa subellipsoidea. Algal Research, 10, 110–120.
G. Breuer et al. (2012). The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresource Technology, 124, 217–226.
Valledor et al. (2014). System-level network analysis of nitrogen starvation and recovery in Chlamydomonas reinhardtii reveals potential new targets for increased lipid accumulation. Biotechnology for Biofuels, 7:171.
S. Zhu et al. (2014). Metabolic changes of starch and lipid triggered by nitrogen starvation in the microalga Chlorella zofingiensis. Bioresource Technology, 152, 292–298.
J. Jia et al. (2015). Molecular mechanisms for photosynthetic carbon partitioning into storage neutral lipids in Nannochloropsis oceanica under nitrogen-depletion conditions. Algal Research, 7, 66–77.
S.K. Lenka et al. (2016). Current advances in molecular, biochemical, and computational modeling analysis of microalgal triacylglycerol biosynthesis. Biotechnology Advances, 34, 1046–1063
A.J. Klok et al. (2013). A model for customising biomass composition in continuous microalgae production. Bioresource Technology, 146, 89–100.
H. Abedini Najafabadi et al. (2015). Effect of various carbon sources on biomass and lipid production of Chlorella vulgaris during nutrient sufficient and nitrogen starvation conditions. Bioresource Technology, 180, 311–317.
Yu et al. (2011). Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microbial Cell Factories, 10:91.

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