Gulo Gene (Marie Bougerol)

In this project I want the bacteria to be able to synthesize L-gulonolactone thanks to the gulo gene. L-gulonolactone is an enzyme that produces Vitamin C. With this enzyme we will be able to synthesize Vitamin C.

It’s a very important enzymatic factor for our body. Vitamin C is used in the synthesis of collagen, red blood cells, contributes to the immune system and plays a role in the metabolism of iron. However, 63 million years ago with the human evolution we lose the gulo gene. Today our body doesn't synthesize L-gulonolactone, so we are depending on dietary Vitamin C. Therefore, it's important to be able to synthesize it.

I use the T7 promoter present in the plasmid chosen, this is the strongest inducible promoter. The T7 RNA polymerase binds to the promoter and starts the transcription. But this system needs to have P_lacUV5-T7 RNA polymerase gene in E. coli. So, we use a BL21(DE3) competent E. coli. Then the transcription stops thanks to the T7 terminator which is after the gulo gene. The L-gulonolactone is secreted in the growth media and is purify with a Ni-NTA chromatography. Thanks to the six histidines tag at the end of the gulo gene sequence, it binds to the Ni-NTA resins. Here is the final sequence of the construct.

When the transformation step into E. coli is done, we select the cells who have incorporated the plasmid. To do it I put the antibiotic Kanamycin in the media, because the plasmid has a KanR gene which provide the resistant of antibiotic.

The gene is cloned to pET-28(+), here is the original sequence. The sequence of the plasmid was download from SnapGene. It is distributed by Novagen. The plasmid provides T7 promoter, kanamycin resistant gene, histidine tag and T7 terminator.

For the cloning strategy I choose to do a Gibson assembly. The steps make possible to obtain a large amount of bacteria with the plasmid and our gene in it. From those colonies we will have L-gulonolactone.

The gene comes from a mammal, so it has introns that disturb the reading frame. That’s why I need to do a reverse transcriptase, the gene will have only the exons. Then I use the superscript series II from Invitrogen as reverse transcriptase. At the end of the protocol I have the cDNA of gulo gene, and I will do a PCR amplification.

An oligo(dT) primer anneals on the poly(A) tails of mRNA. With this primer the m RNA will be reverse transcribed. This are the steps for the reverse transcription that the supplier provides with his kit, you can see it here.

1.In a tube we put:

1µL of oligo(dT) (500µg/mL)

1 µL of L-gulonolactone total RNA at 1µg

1 μL dNTP Mix (10 mM each)

12 µL sterile, distilled water

2. During 5min heat mixture to 65°C and put it quickly in ice. Then we do a centrifugation and get our mixture and put:

4 µL of 5X First-Strand Buffer

2µL of 0.1 M DTT

1µL of RNaseOUT™ (40 units/µL) (optional)

3. Gently mix the products and incubate for 2 min at 25°C.

4. Put 1 µL of SuperScript™ II RT. Mix gently the solution with the pipetting technique and incubate the solution at 25°C for 10 min.

5. Incubate at 42°C for 50 min.

6. And inactivate the reaction by heating at 70°C for 15 min.

I have now the Gulo cDNA and the RNA, to remove RNA I need to add 1 µL of E. coli RNase H and incubate at 37°C for 20 min. Now I can use our cDNA as a template for the PCR.

Gulo is amplified thanks to forward primer gene and ​ reverse primer gene to get a PCR product of Gulo. Gulo Forward primer has a melting temperature of 60.7°C to the matching cDNA sequence. Gulo Reverse primer has a melting temperature of 58°C. I have the Tm thanks to the default parameters of Primer3. Then I add 20 base pair who match to the plasmid, this will allow the gene to anneal to the plasmid. The amplicon is more than 1 kb, so the extension time will be 60 seconds. The recommended annealing temperature thanks to the calculator is 61.5°C. The extension step is around 72°C for 10 minutes, according to the supplier’s site.

Here is the guideline of the PCR with cDNA in PCR using a Taq DNA polymerase.

1.We need to add to the PCR tube:

5 µL of 10X PCR Buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl]

µL of 50 mM MgCl₂

1 µL of 10 mM dNTP Mix

1 µL of Forward primer (10 µM)

1 µL of Reverse primer (10 µM)

0.4µL of Taq DNA polymerase (5 U/µL)

2 µL of cDNA from the reverse transcription reaction

50µL of autoclaved, distilled water

2.Mix gently and layer with 1–2 drops (~50 μL) of silicone oil.

3.Heat reaction to 94°C for 2 min to denature.

4.Perform 15 to 40 cycles of PCR.

Then I do a gel electrophoresis on an agarose gel. I expect to have one single 1355 bp band. The reaction is then clean with a silica column.

The gene is insert in the MCS of the plasmid. To do it we use the Gibson assembly. I design the primers of the plasmid and the insert. They need to have a Tm around 60°C. For the primers of the gene we add 20 base pairs matching to the plasmid. Then I amplify the primers as you saw in the PCR amplification. For the Gibson assembly, 3 enzymes are needed:

The T5 exonuclease degrades the double stranded DNA from the 5’ end. This will expose

Phusion polymerase extend the 3’overhang

Taq DNA ligase joins the two ends of DNA, it operates only at higher temperature

To make the Gibson assembly I put in a tube:

5µL Insert DNA at 5ng/µL

5µL plasmid DNA (pET 28 (+)) at 20ng/µL

10µL Gibson Assembly 2X Master Mix

And then I incubate the tube at 50°C for 15 minutes and the next step is to transform into E. coli.

I'm going to do a heat shock transformation and to put the plasmids into competent DH5-alpha cells obtained from New England Biolabs.

Here is the protocole :

For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.

For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.

Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.

Place on ice for 5 minutes. Do not mix.

Pipette 950 µl of room temperature SOC into the mixture.

Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37°C.

Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

We can't do a blue-white selection because pET-28(+) doesn't have the LacZ. I'm using a Sanger sequencing with individualy screened colonies. Then, the cells grow on LB agar plates containing Kanamycin. After I purify the plasmids using a silica column.

Because gulo gene has less base pair than 1800, the T7 promotor and T7 terminator can cover the whole sequence.

I'm using the BL21(DE3) Competent E. coli cells from NEB.Here is the step for the transformation, it is proceed to a heat transformation:

For (C2527H) Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 minutes.

Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.

Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.

Place on ice for 5 minutes. Do not mix.

Pipette 950 µl of room temperature SOC into the mixture.

Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37°C.

Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24–36 hours or at 25°C for 48 hours.

Then, I place the cells on a selectable media, with Kanamycin and it will growth over night.