Part:BBa_K3370001

Harmonized Gloeobacter rhodopsin (GR) with linker and GFP

Introduction



Gloeobacter rhodopsin introduction

  GR is a light-driven proton pump that originates from the primitive cyanobacteria, Gloeobacter violaceus. It is a seven helix membrane protein located in the inner membrane. Acting as a light-driven proton pump, GR can transfer protons from the cytoplasmic region to the periplasmic region following light absorption. That is, it establishes the proton motive force to push ATP synthase transforming solar energy into universal energy currency, ATP. The reason that GR has a function with light is its specific chromophore, all-trans-retinal. It changes its conformation when induced by light, resulting in a series of protonated and deprotonated reactions on the several amino acids in GR and causing the transfer of protons.

Figure 1: The protein structure of GR-GFP

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.

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_K3370001

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^[2]. 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

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:Proton pumping activity 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

Photototrophic Effect-Growth Measurement

   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

(A)Sodium Azide

   We used sodium azide to block the electron transport chain, and assumed the ATP-producing system will be seriously influenced.(More information is in DESIGN) We measured the growth curve to know at light and dark condition, how sodium azide affects GR-expressing E. coli. We found that although it the growth rate of GR-expressing E. coli is also reduced, we discovered that GR really help producing additional ATP for E. coli to use.

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Figure 6: 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 7: 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


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 8: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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
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• 21
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• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 609
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1571