Team:NYMU-Taipei/Nitrogen starvation
With the development of global economy in the latest demands of energy in world, an average Taiwanese person 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 to fossil fuel to reduce the current energy crisis. In our lab, we used 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 culture systems: open-pond and closed bioreactors. While open-ponds cost way lower than closed bioreactors, open-pond-cultivated 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.
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 accumulation may be lethal to E. coli But the amount of microalgae that contains nitrate and/or 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 the E.coli to prevent contamination. So we planned 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. Then, 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 the suicide of the E.coli at the exact time we want.
NrtA Expression
We successfully transformed NrtA gene from cyanobacteria Synechocystis sp. PCC 6803 to E.coli.
This figure shows restriction enzyme check electrophoresis result of NrtA construct. We use XhoI and HindIII to digest the plasmid, and the expected lengths are 892 bp, 1169 bp, 1540 bp. M represents 1 Kb marker, and the result shows that numbers 2, 4, 6, 8, 10, 12 are correct.
Endolysin Construct
We successfully constructed J23106-B0034-Endolysin-B0010-B0012. The endolysin in this part is from iGEM released part, BBa_K112806, which is the endolysin from the enterobacteria phage T4. Besides the endolysin, in this composite part, we choose BBa_J23106 as the constitutive promoter, BBa_B0034 as the ribosome binding site, BBa_B0010 and BBa_B0012 as the double terminator, all of which are widely used parts in iGEM.
This figure is the gel electrophoresis result of endolysin PCR product. The marker is 100 bp. The expected length is 514 bp.
This figure is the gel electrophoresis result of endolysin backbone PCR product. The marker is 100 bp. The expected length is 2268 bp.
Holin Construct
We successfully constructed R0010-B0034-Holin-B0010-B0012. To control the precise suicide timing, we choose a lactose-inducible promoter, BBa_R0010, to regulate this suicide mechanism. Besides holin coding sequence and the 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 the gel electrophoresis result of holin PCR product. The marker is 100 bp. The expected length is 657 bp.
This figure is the gel electrophoresis result of holin backbone (from BBa_J04450) PCR product. The marker is 1 Kb. The expected length is 2458 bp.
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 regulator.
This is the restriction enzyme check gel electrophoresis result of holin-endolysin construct. The marker is 1 Kb. We used XmaI to digest the sample. The expected total length is 3843 bp.
Endolysin-Holin-NrtA Construct
We successfully constructed R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012-J23118-B0034-NrtA-B0015. This part contains holin, endolysin, and NrtA and has nitrate-capturing function and suicide function.
This figure is the restriction enzyme check gel electrophoresis result of endolysin-holin-NrtA construct. We used XmaI to digest sample 1~3. The result showed that 2 and 3 are correct. M represents 1 Kb marker.
*See our parts: click
*See our experiments protocols: click
Growth Curve Measurement
In order to determine the correct timing to co-culture our modified E.coli with Chlorella vulgaris, we evaluated growth curve of Chlorella vulgaris. We measured the OD value at 680nm by using the spectrophotometer and plotted the growth curve with R software.
However, we encountered some difficulties such as irregular measurement time and personal errors. Therefore, we decided to search for a 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 values from microalgae cultures.
- The above line chart presents growth curve of Chlorella vulgaris. This measurement lasted more than 216 hours and the picture roughly meets our expectation.
- Although the later part of growth curve shows violent fluctuating data, it may be due to effects from environmental nitrogen metabolites of Chlorella vulgaris.
- The measurement result helps us determine the lipid production duration of Chlorella vulgaris, and the timing to add NrtA-transformed E. coli into the medium.
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 measured two different kinds of microalgae during cell growth in the same culture medium, BG-11, respectively.
The photo-bioreactor was designed by Professor Yang. It was made with Arduino and some circuit components. Yang’s students also helped us assemble the device and taught us how to use this device. The photo-bioreactor itself can detect multiple units of organisms at the same time, it has pumps, fans, stir bars and some light bars. In Yang’s laboratory, we created a calibration curve for correcting and we 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.
We expect the engineered E.coli with NrtA gene could express NrtA protein. When we co-cultured 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 whether NrtA protein could capture nitrate and nitrite or not, and then, second, we need to test whether the engineered E.coli could decrease nitrate and nitrite in the medium.
NrtA Protein
To test whether NrtA protein could capture nitrate and nitrite, we used French Pressure Press to lyse the control group (Negative control: CC) and the NrtA-expressing group, after dialysis in BG-11 medium. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration in the medium with/without 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: NrtA-expressing cell.
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: NrtA-expressing cell.
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 BG-11 medium submerged several hours with/without the NrtA-engineered E.coli.
Here are our results.
Figure 2. Nitrate concentration of cell.
Blank: nitrate concentration assay kit assay buffer.
BG11: microalgae culture medium buffer.
CC: Supernatant of BG-11 submerged competent cell.
NrtA: Supernatant of BG-11 submerged NrtA-engineered E.coli.
Table 2. Dunnett’s T3 test of nitrate concentration of cell.
Blank: nitrate concentration assay kit assay buffer.
BG11: microalgae culture medium buffer.
CC: Supernatant of BG-11 submerged competent cell.
NrtA: Supernatant of BG-11 submerged NrtA-engineered E.coli liquid culture.
The nitrate and nitrite concentration of NrtA and competent cell has slightly but not significant difference. The results imply that NrtA protein could capture nitrate and nitrite while the engineered NrtA-expressing E.coli cannot. The engineered E.coli with NrtA gene can express NrtA protein, while NrtA protein might be in the cytosol, so nitrate and nitrite concentration in the medium cannot be decreased. To make NrtA protein become a secreted protein, we are trying to construct signal peptide sequence into it, so that NrtA protein can be released to the medium.
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 tested our suicide mechanism by adding different concentrations 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 bacteria at different time points after the lactose is added.
We measured the OD600 value of the sample, and then aliquot liquid culture into several cuvettes. Next, we added a different amount of 0.5 mM lactose into cuvettes to form different concentrations of lactose, and measured 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 helped us 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 the relative absorbance.
We need to mention that there are some differences in the process of team TAS Taipei and our functional tests. We used different medium to cultivate the bacteria. For the bacteria with holin-endolysin construct, we used Luria-Bertani (LB) broth with 0.9% glucose to cultivate; team TAS Taipei cultivates the bacteria with holin-endolysin-NrtA construct with only Luria-Bertani (LB) broth. According to our previous experimental results, the bacterium doesn't seem to grow well in only LB broth medium. Therefore, to make the figure look similar, team TAS Taipei normalized their data (absorbance at 0 hour is the background). Despite the culture medium difference, the results both show that our suicide mechanism can work. (see more detail about collaboration)
