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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.

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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 our target gene was correctly cloned into the E. coli Lemo21 (DE3).

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Figure 1:Colony PCR result of toxin genes after cloning into E. coli BL21(DE3) ccdB* BBa_K3256440

  Figure 1 was the electrophoresis results of the colony PCR with a marker on the left side and the target gene on the right side. The lengths are labeled beside each band. As a result, we successfully cloned Harmonized GR with GFP linker genes into E. coli.

Functional Test

   After confirming the cloning of target genes, we tested their function by measuring the O.D. value after IPTG induction when the O.D. value reached 0.3 and compared with the O.D. value of the control. The O.D. values were documented in every five minutes for seven hours in total. All experiments were completed in triplicates. (Figure 2~6)

Figure 2:Growth curve of E. coli BL21(DE3) with 500uM IPTG induction ccdB toxin gene (blue), and control (orange)

Calculating Toxicity of Toxin Genes

  We first fit the control’s experiment data to the following equation, dBT/dt=g⋅BT(1−BT/BMax), and we fit the induced data to another equation, dBT/dt=g⋅BT(1−BT/BMax)−Ttoxin⋅BN⋅[toxin]. Next, we compared the two equations to calculate Ttoxin, the toxicity of the toxin gene (Figure 3). In the end, we chose the ydfD gene, which had the most significant toxicity, and the ccdB gene, which had relatively weak toxicity, moving on to the Functional test with mutagens.

Figure 3:Toxicity of different toxin genes. Ttoxin: The toxicity of toxin gene.

Sequence and Features