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.

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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 Lemo21 (DE3) BBa_K3370001

  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.

Protein Expression

  Express Harmonized GR in pET32a various L-rhamnose concentrations From our experimental design, we have tested different L-rhamnose concentration for the best culture environment for GR-expressing system, and we visualized the expression with GFP. We found out that L-rhamnose isn’t needed for GR-expressing E. coli which can also make us know the expression of GR isn’t as hard as we have thought.

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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.

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Figure2:Proton Pumping Activity of Harmonized GR in pET32a expression system From the chart, we can see that when we turned on the light at 420 second, GR pumped proton and changed the pH value outside making the current through the solution increased. When we turned off the light at 540 second, proton flowed back into the cell gradually, pH value increased and the current went through the solution decreased.

Photototrophic Effect-Growth Measurement

   We measured the growth of GR-expressing E. coli to test the function of our GR expression system.

(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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Figure3:Phototrophic growth measurement of GR-expressing E. coli

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Figure4:Phototrophic growth measurement of GR-expressing E. coli under sodium azide(0.001%).

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Figure5:Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition.

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Figure6: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).

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(B)Glucose Consumption

   We used low glucose addition M9 minimal medium to express GR and we wanted to know how GR-expression E. coli consumes GR to gain additional ATP. We measured the glucose consumption to know at light and dark condition, how much glucose be consumed. We found that GR-expressing E. coli consumed GR faster, and we successfully built a system for determining, analysis and prediction for the growth curve with the glucose concentration we have inputted into our culture condition. Glucose Consumption of E. coli WT & GR-expressing E. coli Glucose concentrations are precisely measured in our experiments. We found out that in both light and dark conditions, GR-expressing E. coli consumes glucose much faster than E. coli without GR-expressing system. Therefore, we knew that faster growth of GR-expressing >i>E. coli</i> results from additional ATP produced by GR and faster glucose consumption..

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Figure7: Glucose Consumption of GR-expressing E. coli From Figure 7, we can see that E. coli with GR used up 22.2mM glucose in 12 hours, and E. coli without GR used up in 14 hours. We found out that GR-expressing E. coli consumes glucose much faster than E. coli without GR-expressing system.

Protein Expression Enhancement

   RFP Expression in GR-expressing Lemo21 We expressed RFP in the GR expression system to know how GR-expressing system helps the protein expression.

Co-transform BBa_J04450 in psB1K3 & Harmonized GR with GFP linker in pET32a

   We conducted colony PCR to verify that BBa_J04450 and Harmonized GR were both correctly cloned into the E. coli Lemo21 (DE3).

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Figure8:Colony PCR of co-transform of BBa_J04450(1382 b.p.) & Harmonized GR(2680 b.p.) into E. coli Lemo21

RFP Expression in GR-expression Lemo21

   We conducted colony PCR to verify that BBa_J04450 and Harmonized GR were both correctly cloned into the E. coli Lemo21 (DE3).

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Figure9 Figure19: RFP expression in GR-expressing E. coli (*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001) There is 27% increased protein expression when we incorporate target proteins into GR-expressing E. coli

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
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• 21
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• 23
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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