Part:BBa_K3370601

T7 promoter + LacO + RBS + Harmonized GR with linker and GFP + 6x His-tag + Terminator

This composite part is regulated by a T7 promoter. A polyhistidine-tag is used for purification. In order to assess the expression of GR, we used a linker sequence in conjunction with GFP.

Introduction



Gloeobacter rhodopsin introduction

  Gloeobacter rhodopsin, also known as GR is a seven α-helices transmembrane protein located in the inner membrane. GR is a light-driven proton pump which originates from primitive cyanobacteria, Gloeobacter violaceus. It functions as a proton pump which can transfer protons from the cytoplasmic region to the periplasmic region following light absorption.

Modifications of GR for better folding & expression

  Harmonized GR is different from the common GR. It's been treated under harmonization, one kind of codon optimization. Since the codon frequency of GR in wild-strain and our host-strain is different, we use harmonization, which is an algorithm, to optimize our sequence of codons but without changing the sequence of amino acids.

GFP linker vs. Correct Protein Folding

  The linker is Gly and Ser rich flexible linker, GSAGSAAGSGEF, which provides performance same as (GGGGS) 4 linker, but it doesn’t have high homologous repeats in DNA coding sequence. Therefore, if GFP expresses well, we can ensure that GR proteins fold robustly and are fully soluble and functional. Furthermore, flexible linker could keep a distance between functional domains, so GFP wouldn’t interfere the function of GR.

Figure 1: The protein structure of GR-GFP

Results

Cloning

   We conducted colony PCR to verify that harmonized GR-GFP was correctly cloned into the E. coli Lemo21 (DE3).

Figure 2:Colony PCR result of toxin genes after cloning into E. coli Lemo21 (DE3) BBa_K3370601

Protein 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. 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. We found out that GFP expressed best without L-rhamnose inhibition . Accordingly, Gloeobacter rhodopsin can be easily expressed with proper folding after sequence harmonization, which is good news for GR expression.

Figure 3: Expression of GR with various L-Rhamnose concentrations

Functional Test

   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 Pump Activity Measurement

   We measured the proton pumping amount of Gloeobacter rhodopsin by detecting the photocurrent under intervals of light and dark conditions. Gloeobacter rhodopsin expressing E. coli showed a significant increase in photocurrent under light excitation, compared with the vector control, thus proving its proton pumping activity.

Figure 5: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) 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

Figure 6:Validation of Proton Pumping Activity in GR-expressing E. coli

Photototrophic Effect-Growth Measurement

   The effect of additional ATP increase should affect the behavior of proton pumping expressing E. coli, either in causing growth perturbation or phototrophic growth. We observed the growth of GR-GFP expressing E. coli with beta-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.7 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 7:Phototrophic growth measurement of GR-expressing E. coli

(A)Sodium Azide

   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.

   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, which proved the hypothesis we proposed. This experiment of growth measurement was performed simultaneously with growing conditions without sodium azide, 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.

Figure 8: Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition

Figure 9: Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition at 20th hour(*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001).

(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 use DNS reagent to determine the glucose concentration.

Figure 9: Glucose Consumption of 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(Q_Max) 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

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-expression 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 9:RFP expression in GR-expressing E. coli (*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001)
GR-expressing E. coli shows stronger fluorescence intensity than the vector control ones.

Figure 10:Visualization of RFP Expression

Sequence and Features

Assembly Compatibility:

• 10
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• 12
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• 23
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• 25
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  Illegal NgoMIV site found at 689
  Illegal NgoMIV site found at 1764
  
• 1000
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  Illegal BsaI.rc site found at 1651