Team:NCTU Formosa/Results

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Overview
To incorporate light driven proton pump into E. coli, we first built up the gene construct and expressed the transmembrane protein and observed its influence exerted on E. coli to evaluate its function and finally its ability to enhance target protein expression [1] (Graph 1).
Graph 1: Experiment workflow
Gene Construct & Protein Expression
Overall, we constructed three gene types in E. coli, Lemo21(DE3) in the experiment(Graph 2). We did colony PCR and digest to check their genotype respectively.
Graph 2: Light driven proton pump expressing E. coli (left). Chromophore producing E. coli (middle). Target protein expressing E. coli (right).
Harmonized GR Expression
(A) Harmonized GR in pET32a Cloning PCR & Digest
We conducted colony PCR to verify that harmonized GR-GFP was correctly cloned into the E. coli Lemo21 (DE3).
Figure 1: Colony PCR result of harmonized GR-GFP after cloning
into E. coli Lemo21(DE3). (2680 b.p.)
(B) Harmonized GR Expression Tests
Expression of harmonized GR-GFP in pET32a with various L-Rhamnose concentrations
The proper folding of transmembrane light-induced proton pump(GR) can be visualized by GFP. (More information see Design) It is generally acknowledged that transmembrane proteins are difficult targets for expression, so we chose E. coli, Lemo-21, which features tunable T7 promoter expression system for the expression of GR[2]. We found out that GFP expressed best without L-Rhamnose inhibition (Fig. 2a, 2b). Accordingly, Gloeobacter rhodopsin can be easily expressed with proper folding after sequence harmonization, which is good news for GR expression.
Figure 2(a): GR-GFP expression with various L-Rhamnose concentrations
Figure 2(b): The best expression occurred without addition of L-Rhamnose.
BBa_K274200 Cloning & Expression
(A) CrtEBIY in psB1K3 Cloning PCR
We conducted colony PCR and digest to verify that BBa_K274200 was correctly cloned into E. coli Lemo21 (DE3).
Figure 3: Confirmation of BBa_K274200 by digest(4555 b.p./ 2079 b.p.)
Figure 4: Colony PCR of BBa_K274200(CrtEBIY), E. coli Lemo21(5078 b.p.)
(B) β-carotene Extraction in acetone
The chromophore we used in GR-GFP was β-carotene, the precursor of all-trans retinal, which could be produced by the iGEM biobrick BBa_K274200. Due to its difficulty in producing all-trans retinal in E. coli, we tried β-carotene as an alternative chromophore. We thus produced β-carotene by ourselves and extracted it for extracellular addition in the subsequent functional test experiment.
We extracted β-carotene from CrtEBIY expressing E. coli and measured the spectrum to confirm if β-carotene be successfully produced.
β-carotene extraction after consulting Professor Kao
After consulting Professor Kao in NCTU DBT to troubleshoot our β-carotene extraction method (More information is in Human Practices), we have confirmed that β-carotene is successfully produced (Fig. 5) in E. coli with the new protocol (More information is in Protocol).
TAS_Taipei validation of β-carotene extraction in acetone
We collaborated with TAS_Taipei for validation of β-carotene extraction. (More information is in Collaborations) They followed the protocol (More informations is in Protocol) we constructed, and successfully repeated the experiment (Fig. 6).
Figure 5: β-carotene Extraction Absorption Spectrum
Figure 6: TAS_Taipei Validation(green line): β-carotene extraction absorption spectrum
GR Functional Tests
After the expression of GR in E. coli, we tested the light-induced proton pump by measuring photocurrent and evaluated the influence on bacterial growth.
Proton Pumping Activity Measurement
With an aim to directly observe the proton pumping activity of GR-GFP in living cells, we consulted senior iGEMer, Lung-Chieh Chen, and instructor, prof. Wen-Liang, Chen. The lab offered its special techniques originally used as an electrochemical biosensor for detecting various biomolecules, which features its ability to accurately measure the amount by the detecting the resistance or electric current[3]. We thus designed an experiment based on this platform by directly depositing a droplet of living GR-expressing E. coli on the electrode of the detection chip. The droplet served as an bridge between the two electrodes. By detecting the difference of current produced during the experiment, we hypothesized that a difference of current induced by illumination, which could be viewed as photocurrent produced by the proton pump.
We measured the proton pumping amount of Gloeobacter rhodopsin by directly detecting the electric current under intervals of light and dark conditions.(The arrangement of the experiment see Fig. 7, and the measuring detail see Protocol) Gloeobacter rhodopsin expressing E. coli showed a significant increase in photocurrent under light excitation (Fig. 8), compared with the vector control, thus proving its proton pumping activity.
Figure 7: Photocurrent Measurement Device
Figure 8: Photocurrent Measurement of GR-expressing E. coli
The proton pumping efficiency was determined by the increase in photocurrent at the duration of illumination. We considered the first illumination to be the genuine representation of reflecting the proton pumping activity of GR, so we took the first duration (420 sec to 540 sec, Fig. 9) and analyzed it through proton pumping simulation, and the proton pumping of GR was 0.16 (extracellular, ΔH+ × 10 -7/min OD), whereas the value of GR’s proton pumping rate by Pil Kim et al was 0.38[5].
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Figure 9: Validation of Proton Pumping Activity in GR-expressing E. coli
Phototrophic Effect-Growth Measurement
The effect of additional ATP increase should affect the behavior of proton pumping expressing E. coli, either in causing growth perturbation[4] or phototrophic growth [5]. We observed the growth of GR-GFP expressing E. coli with β-carotene as chromophore and further evaluated its potential role in chemiosmotic effect. We used modified M9 minimum medium to evaluate the phototrophic growth.
The whole incubation process was illuminated by white light LED strip and the O.D.600 was documented every 20 minutes in either transparent or dark 96-well plates by LogPhase 600 for 22 hours. Fig.10 showed that GR-expressing E. coli showed better growth rate than vector control once. The maximum cell growth of GR-expressing E. coli is 0.071(O.D.600/h), whereas its pET32a control is 0.054 (O.D.600/h).
Figure 10: Phototrophic growth measurement of GR-expressing E. coli
(A)Sodium Azide
Graph 3: Hypothetical mechanism of the role of light induced proton pump(GR) in E. coli
To further investigate the role of GR-GFP expressed in E. coli, we added sodium azide to inhibit the respiratory electron transport chain to assess the function of GR-GFP. We hypothesized that GR-GFP’s proton pumping activity could compensate for the loss of function of respiratory electron transport chain due to sodium azide as graph 3 shows[6].
Figure 11: Phototrophic growth measurement of GR-expressing
E. coli under sodium azide(0.001%)
Figure 12: Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition
Figure 13: Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition at 20th hour
(**:p value<0.01/
****:p value<0.0001)
With the addition of sodium azide, it strongly inhibited the growth of E. coli; moreover, we found that GR-expressing E. coli survived in 0.01% sodium azide growing environment (Fig. 11), which proved the hypothesis we proposed. This experiment of growth measurement was performed simultaneously with growing conditions without sodium azide (Fig.10). Altogether, we found that the proton pump we expressed in E. coli serves as an alternative for respiratory electron transport chain, thus proving its function of creating proton gradient in E. coli. The experiment was done with 3 technical replicates and the growing pattern shows significant difference among GR-expressing E. coli and vector control (Fig. 13).
(B)Glucose Consumption
With respect to the phototrophic growth pattern observed, faster growth of GR-expressing E. coli not only relies on the proton gradient, additional ATP it produces, but also on its carbon sources, mass increase, for growth. We were next interested in finding the consumption rate of glucose in GR-expressing E. coli. Basically, we expected the higher consumption rate of glucose with additional ATP produced by GR. We used M9 medium with glucose (0.4%, 22.2mM), and used DNS reagent to determine the glucose concentration.
Glucose Measurement Standard Curve
Before we measured the glucose concentration in our M9 minimal culture, we should set up a standard for it. The R square of our standard curve is 1, so we ensured that the glucose concentrations were precisely measured.
Figure 14: Glucose Measurement Standard Curve
Glucose Consumption of E. coli -WT & GR-expressing E. coli
We found that GR-expressing E. coli consumed GR faster, as it exhausted glucose in 12 hours, while the vector control one (pET32a, Lemo21) took 14 hours for glucose depletion(Fig.15). The maximum glucose uptake rate(QMax) of GR-expressing E. coli Lemo21 is 11.28(Mm/O.D.600·h) whereas that of vector control one is 9.47(Mm/O.D.600·h). Also, we successfully built a system for the prediction for the growth curve with glucose concentration, we have integrated it into our culture condition optimization model. (More information is in ENGINEERING)
Figure 15: Glucose Consumption of GR-expressing E. coli
Summary
In the functional test of GR-GFP, we provided evidence for proving its pumping activity and its participating role in E. coli’s chemiosmotic proton gradient with the sodium azide test. The proton pumping activity was first measured by the chip in photocurrent difference with illumination, and its phototrophic growing effect was revealed by growth measurement with our proton pump with faster glucose consumption, which was in line with our expectation that GR-GFP offers an additional facilitating source of energy. In that GR-GFP gene can be easily expressed and visualized in E. coli, it may be also expressed in other strain of E. coli and modified through extensive gene engineering techniques.
Protein Expression Enhancement
Now that we had proved the gene to be functional and beneficial to E. coli, we finally came to the final proof of concept in the experiment. The concept of E. hybrid can be exemplified with a simple target protein, red fluorescence protein, RFP. We assessed the ability of engineered GR-GFP expressing E. coli, Lemo21 by expressing RFP. We expected the fluorescence intensity to be stronger than vector control ones, if so proving its ability to achieve the goal of protein expression enhancement and its protein function.
RFP Expression in GR-expressing Lemo21
We expressed RFP in the GR-expressing system to know how it helps the protein expression.
(A) RFP(BBa_J04450) in psB1K3 Cloning PCR
We conducted colony PCR to verify that BBa_J04450 and harmonized GR-GFP were both correctly cloned into the E. coli Lemo21 (DE3).
Figure 16: Colony PCR result of BBa_J04450(1382 b.p.)
Figure 17: Colony PCR of co-transform of BBa_J04450(1382 b.p.) &
harmonized GR(2680 b.p.) into E. coli Lemo21
Figure 18: Colony PCR of co-transform of BBa_J04450(1382 b.p.) &
pET32a(754 b.p.) into E. coli Lemo21
(B) RFP Expression in GR-expressing E. coli Lemo21
We cultivated both the GR-expressing E. coli and vector control ones in LB with IPTG induction in LB broth for incubation. We measured the end point of the final samples and compared their RFP fluorescent intensity.
Figure 19(a): RFP expression in GR-expressing E. coli The test was done with triplicate(*:p value<0.05)
Figure 19(b): Visualization of RFP Expression
GR-expressing E. coli shows stronger fluorescence intensity than the vector control ones (Fig. 19a, 19b), as can be visualized under blue light illuminator (BLooK, GeneDireX).
Reference
  1. Kim, H.-J., et al. (2012). "An engineered Escherichia coli having a high intracellular level of ATP and enhanced recombinant protein production." Applied microbiology and biotechnology 94(4): 1079-1086
  2. Schlegel, S., et al. (2012). "Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21 (DE3)." Journal of molecular biology 423(4): 648-659.
  3. Chen, L.-C., et al. (2020). "Improving the reproducibility, accuracy, and stability of an electrochemical biosensor platform for point-of-care use." Biosensors and Bioelectronics 155: 112111.
  4. Na, Y.-A., et al. (2015). "Growth retardation of Escherichia coli by artificial increase of intracellular ATP." Journal of industrial microbiology & biotechnology 42(6): 915-924.
  5. Kim, H. A., et al. (2017). "An evolutionary optimization of a rhodopsin-based phototrophic metabolism in Escherichia coli." Microbial cell factories 16(1): 111
  6. Walter, J. M., et al. (2007). "Light-powering Escherichia coli with proteorhodopsin." Proceedings of the National Academy of Sciences 104(7): 2408-2412.
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