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 PCR product.
The marker is 1kb. The length is 705bp 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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