Team:Sorbonne U Paris/Contribution



In order to validate the bronze criterion, we decided to characterize a total reporter gene: BBa_I13521, a Ptet R promoter which includes PtetR-RBS-mRFP-dTerminator.


Experiments were carried on DH5 alpha bacteria without artificial addition of the tetR repressor system.


Day 1: The DNA corresponding to this part is amplified and extracted from liquid cultures of bacteria thanks to the Promega extraction kit. Control digests by Eco RI and Pst I were carried out in order to verify the presence of the part of 923 bp. DNA fragments were analyzed on a 1.2% agarose gel. The 8 clones tested have the correct BBa_I13521.






The plasmid DNAs containing part 1 to 8 are used to transform competent DH5 α bacteria which are then plated on LB medium + Chloramphenicol 34 μg/mL and incubated overnight at 37°C.

Day 2: A clone resulting from each transformation is subcultured in liquid culture of 5 mL of LB medium + Chloramphenicol 20 μg/mL. These cultures are incubated overnight at 37°C with 200 rpm shaking.

Day 3: The 8 liquid cultures are diluted to an OD at 600 nm of 0.1 in LB medium + Chloramphenicol 20 μg/mL.
The liquid cultures are then placed at 37°C, with stirring at 200 rpm until an OD600 of 0.4 is reached.

Each of the 8 liquid cultures is divided into 3, corresponding to 3 different concentrations of tetracycline: 0 ng/mL - 100 ng/mL – 10 000 ng/mL.

A time course experiments is realized, measuring the OD600 and mRFP fluorescence (535 nm (excitation) / 620 nm (emission) of 100µL of each culture every 30min.

As expected, increasing the concentration of tetracycline slowls down the OD 600, meaning that even at this low concentration, the tetracycline is toxic for bacteria. mRFP fluorescence was too low to overpass background signal (data not shown).

To check if bacteria were expressing the mRFP, we observed the cells after 6h incubation at 37°C using an epi-fluorescence microscope. Doing so, we were able to detect mRFP signal, the bacteria being red. Therefore, we quantified the mRFP fluorescence signal for the bacteria transformed with the BBa_I13521 part.




We observed no significative variation of the mRFP signal depending on the tetracycline concentration.
The BBa_I13521 part is described as “Constitutive on mRFP (positive control)” but the subpart description is not coherent with this description, as it is stated that this reporter construct has a ptetR promoter (regulated by tetracycline, thus not constitutive). The two description being incoherent, we wanted to identify the correct one. Our data are in favor of a constitutive expression of the mRFP, not affected by the addition of tetracycline.


We also tried other caracterisations that didn't work.


We previously tried to characterize an other constitutive promoter: BBa_I14033

We Used the 3A Assembly : 3A Assembly




The part A will be digested with Eco RI and Spe I, the part B with Xba I and Pst I and the linearized plasmid with Eco RI and Pst I.
The Spe I site and the Pst I site are compatible, so it will be assembled in the following order : A + B


Bacteria transformation

Material :

  • DH5 α
  • SOC medium
  • Ice
  • Petri plate with LB medium + antibiotic

Transform 10 µL of bacteria with 1 µL of DNA.
Incubate 30 min on ice, then 30 secs at 42°C then back on ice for 2 min.
Add 100 µL of SOC medium
Spread on petri plate in sterile condition


Miniprep protocol

Material :

  • Promega Miniprep kit
  • Centrifuge
  • 2 mL Eppendorf tubes
  • 1,5 mL Eppendorf tubes
  • Void machine
  • Collecting tubes

Centrifugate liquid culture for 10 min at max speed.
Eliminate supernatant.
Add 600 µL H2O to resuspend the pellet and vortex.
Transfer into a 2 mL Eppendorf tube.
Add 100 µL of lyse buffer shake and wait 2 min.
Add 350 µL of neutralization buffer and shake.
Centrifugate for 3 min at max speed.
Put the columns on the void machine.
Transfer supernatant in the columns.
Add 200 µL of wash endotoxin.
Add 400 µL of Wash solution.
Put the columns in the collecting tubes.
Centrifugate the columns 2 min at max speed.
Put the columns in the 1,5 mL Eppendorf tube.
Add 30 µL elution buffer and wait 1 min.
Centrifugate 1 min at max speed.
The plasmid is now in the Eppendorf tube.

We used the RBS BBa_J61100, GFP BBa_E0040 and the Terminator BBa_B0015.
For the first step, we assembled into a kanamycin resistant plasmid (pSB1K3) the Promoter and the RBS in one hand and GFP and Terminator on the other hand. Here the promoter and the GFP are the parts A and the RBS and the Terminator are the parts B. The ligations products are Promoter-RBS (plasmid 1) and GFP-Terminator (plasmid 2).

Digestion

Material :

- CutSmart
- NEB Buffer 2
- Restriction enzymes (Eco RI-HF, Pst I, Pst I-HF, Dpn I, Spe I-HF, Xba I-HF)

▪ Enzyme Master Mix for Plasmid Backbone (25 µL total, for 5 reactions)

  • 5 µL NEB Buffer 2
  • 0,5 µL Eco RI-HF
  • 0,5 µL Pst I
  • 0,5 µL Dpn I (Used to digest any template DNA from production)
  • 18,5 µL dH20

▪ Enzyme Master Mix for Part A (25 µL total, for 5 reactions)

  • 5 µL CutSmart
  • 0,5 µL Eco RI-HF
  • 0,5 µL Spe I-HF
  • 19 µL dH20

▪ Enzyme Master Mix for Part B (25 µL total, for 5 reactions)

  • 5 µL CutSmart
  • 0,5 µL Xba I-HF
  • 0,5 µL Pst I-HF
  • 19 µL dH20

▪ Digest Plasmid Backbone

  • Add 4 µL linearized plasmid backbone (25 ng/µL for 100 ng total)
  • Add 4 µL of Enzyme Master Mix

▪ Digest Part A

  • Add 4 µL Part A (25 ng/µL for 100 ng total)
  • Add 4 µL of Enzyme Master Mix

▪ Digest Part B

  • Add 4 µL Part B (25ng/µL for 100 ng total)
  • Add 4 µL of Enzyme Master Mix

Digest all three reactions at 37°C/45 min, heat kill 80°C /20 min.


Ligation

Material :

- T4 Ligase buffer
- T4 Ligase
- Ice

  • Add 2 µL of digested Plasmid Backbone (25 ng)
  • Add equimolar amount of Part A (Eco RI-HF Spe I digested) fragment (< 3 µL)
  • Add equimolar amount of Part B (Xba I Pst I digested fragment) (< 3 µL)
  • Add 1 µL T4 DNA ligase buffer. Note : Do not use quick ligase
  • Add 0,5 µL T4 DNA ligase
  • Add water to 10 µL
  • Ligate 16°C/30 min, heat kill 80°C/20 min
  • Transform with 1-2 µL of product

In order to assess if we got our fragments in the plasmid, we digested our plasmid with Eco RI and Pst I and migrated in agarose or acrylamide gel to confirm the presence of the part.
Then we assembled the two parts into a pSB1A3 (ampicillin resistant) to get our final construct. Here the plasmid 1 will be the part A and the plasmid 2 will be the part B.
We also make a control construct without the promoter. The RBS (part A) is assembled to the plasmid 2 (part B) to obtain this construct.

Results


We digested our final construct (Fi1-3) with Eco RI and Pst I. We expect to have two signals :
2118 bp and 960 bp for our final construct.
2167 bp and 894 bp for CD6.



The plasmid Fi3 seems to have our construct but we could not see any fluorescence. We suspect that the GFP part does not work. The plasmid contains a double site AlwNI, one into the GFP part, so we digested with AlwNI and we expect to get two bands at 2271 bp and 807 bp. But the electrophoresis did not show the expected profile, as we got only one band. We hypothesized that the GFP part was not the right sequence so we decided to adopt a new strategy.

We have tried again to clone the GFP (BBa_E0040) and double-terminated rrnBT1-T7TE (BBa_B0015) sequences in plasmid pSB1K3 by changing the restriction enzymes, the buffers and the ligase as well as its buffer.

All digestive reactions are performed according to the recommendations given by DoubleDigest Calculator-Thermo Scientific.

Simple or Double digest protocol

Material :

- Plasmid DNA
- Restriction enzyme (Eco RI, Pst I, Spe I, Xba I, Dpn I)
- 10X O, R or Tango Buffer
- H2O

  • 500 ng to 1 μg of DNA
  • 5 to 10 units of enzyme 1
  • (5 to 10 units of enzyme 2)
  • 2 μL of 10X Buffer
  • H2O up to 20 μL

Incubation time: 1 hour
The temperature is enzyme dependent

The fold excess of enzyme, the reaction buffer and the incubation temperature are chosen according to the recommendations given by DoubleDigest Calculator-Thermo Scientific.


Ligation protocol

Material :

- Insert DNA
- Vector DNA
- T4 DNA Ligase
- 10X T4 DNA Buffer
- H2O

  • Insert DNA
  • Vector DNA
  • 5 units of T4 DNA Ligase
  • 1 μL of 10X T4 DNA Buffer
  • H2O up to 10 μL

Incubate overnight at 16/18°C.

The amounts of insert and vector to be used are defined according to the recommendations of NEBio Ligation Calculator.

All plasmid extraction were performed using Promega's PureYield ™ Plasmid Miniprep System.

Plasmid pSB1K3 was digested with Eco RI, Pst I as well as Dpn I in O buffer. The plasmid containing GFP was digested with Eco RI and Spe I in R buffer and the plasmid containing the double terminator was digested with Xba I and Pst I in Tango buffer.
Digestion profile has been checked by a migration on a 1.5% agarose gel showing that GFP and the Terminator where correctly removed from their plasmid.
Ligation was then performed by incubating the digested pSB1K3, GFP Bba_E0040 and dTer BBa_B0015 with T4 ligase in its buffer.

Top 10 competent bacteria were then transformed with the ligation product and then plated on LB + Kanamycin 30 μg/mL medium and incubated overnight at 37°C. The following day, 7 colonies were subcultured in liquid culture of 5 mL of LB medium + Kanamycin 50 μg/mL overnight at 37°C with shaking 200 rpm.


Transformation protocol used for all experiments


  • Add 50 ng of plasmid DNA to 50 μL of DH5 α thermo competent bacteria.
  • Incubate 30 minutes on ice.
  • Heat shock for 45 seconds at 42°C.
  • Back on ice 5 minutes.
  • Add 950 μL of SOC medium to the bacteria.
  • Incubate 1 hour at 37°C with shaking 200 rpm.
  • Spread 100 μL on LB + Chloramphenicol 34 μg/mL. (10%)
  • Centrifuge the remaining 900 μL for 3 minutes at 2500 rpm to pellet the bacteria.
  • Remove 800 μL of supernatant.
  • Resuspend the pellet in the remaining 100 μL and spread on LB + Chloramphenicol 34 μg/mL (90%).
  • Incubate the dishes at 37°C overnight.

The next day the plasmid DNA was extracted from the bacteria using the Promega kit and control digests were performed to check the presence of the GFP-dTer insert. The plasmid DNA is digested with Eco RI and Pst I in an O buffer and the digests are then migrated on 1.5% agarose gel. This digestion allowed us to observe that none of the 7 plasmid DNAs contains the desired insert.

A new strategy is envisaged to characterize the promoter Bba_I14033. We decided to change the reporter gene. We will use BBa_K518012 which included RBS-mRFP-dTerminator. This plasmid will serve as a negative control (without a promoter) and we have to insert our promoter P (Cat) into this plasmid digested by Eco RI and Xba I.

After amplifying the plasmid DNA of our reporter gene BBa_K518012, it is digested with Eco RI and Xba I in Tango buffer. Its extremities were then dephosphorylated by Fast Alkaline Phosphatase (FastAP) in its buffer. The linearized and dephosphorylated plasmid DNA is then eluted on an agarose gel with the GeneJET Thermoscientific Gel Extraction Kit.

The P (Cat) promoter and the plasmid containing the reporter gene are bound by T4 ligase in its buffer. A negative control is performed with only the linearized plasmid containing the reporter gene but without the promoter. The ligation is done overnight at 16/18°C.

DH5α competent bacteria are then transformed by the ligation product, or by the negative ligation control, or only by the plasmid containing the reporter gene, without the P (Cat) promoter. The bacteria are plated on medium LB + Chloramphenicol 34 μg/mL then incubated on the night at 37°C.

The following day, we observed a lot of bacteria in our P (Cat) – mRFP reporter gene dish as well as in the control dish that only contains the circular reporter gene. Finally, we do not find any colony in the negative ligation control dish that contains the digested and dephosphorylated plasmid.

We do not find any expression of the mRFP in the P (Cat) - mRFP reporter gene bacteria, which suggests that the promoter is not active.

We picked 6 colonies of the P (cat) - mRFP reporter gene that we passed in liquid culture of 5 mL of LB medium + Chloramphenicol 20 μg/mL. These cultures are incubated overnight at 37°C with shaking 200 rpm. The next day all the plasmid DNAs are extracted using the Promega extraction kit.