Difference between revisions of "Team:INSA-UPS France/test/description"

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           Then purify the products thanks to PCR purification kit
 
           Then purify the products thanks to PCR purification kit
 
         </li>
 
         </li>
 +
      </ol>
 +
    </section>
 +
 +
    <div class="article_offset" id="prot2"></div>
 +
    <section>
 +
    <h1>Gel extraction</h1>
 +
      <h2>Procedure</h2>
 +
      <p>
 +
        <i>Please, before doing your preparative gel, use one sample to make an analityc one !</i>
 +
      </p>
 +
      <ol>
 +
        <li>Equilibrate a water bath or heat block to 50&deg;C.</li>
 +
        <li>
 +
        Excise a minimal area of gel containing the DNA fragment of interest.<br />
 +
       
 +
          <ul>
 +
            <li><b>Crucial:</b> To protect the UV box, it is a good idea to place the gel on a glass plate if available. </li>
 +
            <li>Try to get as little excess gel around the band as possible.</li>
 +
          </ul>
 +
 +
        </li>
 +
        <li>Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.</li>
 +
        <li>
 +
          Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:
 +
          <table>
 +
            <tr>
 +
              <th>Gel</th>
 +
              <th>Tube</th>
 +
              <th>Buffer L3 Volume</th>
 +
            </tr>
 +
            <tr>
 +
              <td>&le;2% agarose</td>
 +
              <td>1.7 mL polypropylene</td>
 +
              <td>3:1 (i.e., 1.2 mL Buffer L3: 400 mg gel piece)</td>
 +
            </tr>
 +
            <tr>
 +
              <td>&gt;2% agarose</td>
 +
              <td>5 mL polypropylene</td>
 +
              <td>6:1 (i.e., 2.4 mL Buffer L3: 400 mg gel piece)</td>
 +
            </tr>
 +
          </table>
 +
        </li>
 +
        <li>
 +
          Place the tube with the gel slice and Buffer L3 into a 50&deg;C water bath or heat block. Incubate the tube at 50&deg;C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution.
 +
          <ul>
 +
            <li>Note: High concentration gels (&gt;2% agarose) or large gel slices may take longer than 10 minutes to dissolve.</li>
 +
          </ul>
 +
        </li>
 +
        <li>
 +
          After the gel slice appears dissolved, incubate the tube for an additional 5 minutes.
 +
          <ul>
 +
            <li>
 +
              Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
 +
            </li>
 +
          </ul>
 +
        </li>
 +
        <li>
 +
          <b>Before Starting:</b> Add ethanol to the Wash Buffer (W1) according to the label on the bottle.
 +
        </li>
 +
        <li>
 +
          <b>Purifying DNA Using a Centrifuge</b>
 +
        </li>
 +
        <li>
 +
          <b>Load.</b> Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel.
 +
          <ul>
 +
            <li>
 +
              Note: The column reservoir capacity is 850 &mu;L.
 +
            </li>
 +
          </ul>
 +
        </li>
 +
        <li>
 +
          <b>Bind.</b> Centrifuge the column at &gt;12,000 &times; g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
 +
        </li>
 +
        <li>
 +
          <b>Wash.</b> Add 500 &mu;L Wash Buffer (W1) containing ethanol to the column.
 +
        </li>
 +
        <li>
 +
          <b>Remove Buffer.</b> Centrifuge the column at &gt;12,000 &times; g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
 +
        </li>
 +
        <li>
 +
          <b>Remove Ethanol.</b> Centrifuge the column at maximum speed for 1–2 minutes. Discard the flow-through.
 +
        </li>
 +
        <li>
 +
          <b>Elute.</b> Place the column into a Recovery Tube. Add 30 &mu;L Elution Buffer (E5) to the center of the column. Incubate the tube for 1 minute at room temperature.
 +
        </li>
 +
        <li>
 +
          <b>Collect.</b> Centrifuge the tube at &gt;12,000 &times; g for 1 minute.
 +
        </li>
 +
        <li>
 +
          <b>Store.</b> The elution tube contains the purified DNA. Store the purified DNA at 4&deg;C for immediate use or at &minus;20&deg;C for long-term storage.
 +
        </li>
 +
      </ol>
 +
    </section>
 +
 +
    <div class="article_offset" id="prot3"></div>
 +
    <section>
 +
 +
    <h1>Gel migration</h1>
 +
      <h2>Introduction</h2>
 +
      <p>
 +
        This protocol is the classical one used for electrophoresis. - You can adapt the concentration of agar according to the length of your fragment 1% agar if the DNA fragments are big 2% agar if the DNA fragments are small (the bigger fragment are sticked together) - Adapt the volume of the gel 15 to 30 mL for small gels and 150 to 200 mL for big gels
 +
      </p>
 +
 +
      <h2>Procedure</h2>
 +
      <ol>
 +
        <li>
 +
          Thoroughly rinse gel housing and well-comb with dH2O.
 +
        </li>
 +
        <li>
 +
          Place gel mold perpendicular to flow direction, ensuring proper sealing of rubber gaskets.
 +
        </li>
 +
        <li>
 +
          Add the calculated amounts of 0.5xTBE and agarose to a fresh Erlenmeyer flask.
 +
        </li>
 +
        <li>
 +
          Heat in microwave until mixture can be dissolved.
 +
          <ul>
 +
            <li>
 +
              <b>CRITICAL:</b> Do not let the mixture boil over and out of the flask. Typical heating time for 50mL in a 2.45GHz microwave oven at full power is 30s. USE HEAT GLOVES
 +
            </li>
 +
          </ul>
 +
        </li>
 +
        <li>
 +
          Gently swirl until well mixed and gently swirling periodically until ~55&deg;C.
 +
        </li>
 +
        <li>
 +
          Gently pour molten agarose gel into housing, avoiding air bubbles.
 +
        </li>
 +
        <li>
 +
          Place desired well comb in desired position.
 +
        </li>
 +
        <li>
 +
          Once gelled, carefully remove well comb in a uniform fashion.
 +
        </li>
 +
        <li>
 +
          Remove gel mold and place in parallel direction to flow
 +
          <ul>
 +
            <li>
 +
              <b>CRITICAL:</b> the deposit line has to be at the anode (negative pole)
 +
            </li>
 +
          </ul>
 +
        </li>
 +
        <li>
 +
          Fill gel box with 0.5 xTAE until the gel is well covered.
 +
        </li>
 +
        <li>
 +
          Place the ladder on the gel, the native and digested plasmid (write down the gel map)
 +
          <ul>
 +
            <li><b>TIP:</b> When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. </li>
 +
          </ul>
 +
        </li>
 +
        <li>
 +
          Run the electrophoresis for 20-30min at 100V until the dye line is approximately 80% of the way down the gel
 +
        </li>
 +
        <li>
 +
          Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
 +
        </li>
 +
        <li>
 +
          Place the gel into a container filled with 100 mL of TAE running buffer and 5 &mu;L of EtBr, place on a rocker for 20-30 mins, r
 +
        </li>
 +
        <li>
 +
          Place the gel into a container filled with water and destain for 5 mins.
 +
        </li>
 +
        <li>
 +
          Reaveal under UV lamp, visualize your DNA fragments
 +
        </li>
 +
      </ol>
 +
    </section>
 +
 +
 +
    <div class="article_offset" id="prot4"></div>
 +
    <section>
 +
    <h1>Miniprep</h1>
 +
      <h2>Introduction</h2>
 +
      <p>
 +
        This protocol was taken from the <a href="https://tools.thermofisher.com/content/sfs/manuals/MAN0013117_GeneJET_Plasmid_Miniprep_UG.pdf">ThermoScientific GeneJET Plasmid Miniprep Kit</a>. Safety: Both the Lysis Solution and the Neutralization Solution contain irritants. <b>Wear gloves when handling these solutions.</b>
 +
      </p>
 +
 +
      <h2>Procedure</h2>
 +
      <ul>
 +
        <li>
 +
          Note: All steps should be carried out at room temperature. All centrifugations should be carried out in a microcentrifuge at &ge; 12 000 x g (10 000-14 000 rpm, depending on the rotor type).
 +
        </li>
 +
        <li>
 +
          Be sure that the concentrated solutions have been diluted with the appropriated buffer
 +
        </li>
 +
      </ul>
 +
      <ol>
 +
        <li>
 +
          Pick a single colony from a freshly streaked selective plate to inoculate 5mL of LB medium supplemented with the appropriate selection antibiotic.
 +
        </li>
 +
        <li>
 +
          Incubate for 12-16 hours at 37&deg;C while shaking at 200-250 rpm
 +
        </li>
 +
        <li>
 +
          Centrifugate the bacterial culture, &gt;12 000 g in a microcentrifuge for 2 minutes at room temperature. Repeat until there is no more media.
 +
        </li>
 +
        <li>
 +
          Add to the pelleted cells:
 +
          <ul class="classic_ul">
 +
            <li>250 &mu;L of Resuspension Solution and vortex</li>
 +
            <li>250 &mu;L of Lysis Solution and invert the tube 4-6 times. WAIT 2 min</li>
 +
            <li>350 &mu;L of Neutralization Solution and invert the tube 4-6 times.</li>
 +
            <li>Lysis buffer must be neutralized before 5 minutes</li>
 +
          </ul>
 +
        </li>
 +
        <li>Centrifuge 5 minutes.</li>
 +
        <li>Transfer the supernatant to the Thermo Scientific GeneJET Spin Column. Centrifuge 1 minute</li>
 +
        <li>Add 500 &mu;L of Wash Solution and centrifuge for 60 s and discard the flow-through</li>
 +
        <li>Repeat step 5.</li>
 +
        <li>Centrifuge empty column for 1 minute.</li>
 +
        <li>Dry for 5 minutes </li>
 +
        <li>Transfer the column into a new tube.</li>
 +
        <li>Add 30 &mu;L of Elution Buffer to the column and incubate 2 minutes. </li>
 +
        <li>Centrifuge 2 minutes.</li>
 +
        <li>Collect the flow-through.</li>
 
       </ol>
 
       </ol>
 
     </section>
 
     </section>

Revision as of 15:51, 6 October 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Experiments

go to Protocols

E. coli module -- UNDER CONSTRUCTION
V. harveyi as a new synthetic biology chassis
Cloning the engineered receptor
Information transmission: producing diacetyl
Preliminary clonings:
Assembly cloning:
receptor part 1 + 2 + tetR
Culture for diacetyl production and NMR analysis

Ligation, transformation of Vh_quorum

One colony has grown O/N on the Petri dish. This colony was picked in liquid LB media culture with Cm.

Possible explanations for the low amount of colonies:

  • pippeting imprecisions
  • Strong concentration of Cm
  • Competent cells: 106 only

Another ligation was carried out today, using more DNA and another Cm concentration. The ligation was processed as follows: Ligation protocol

A ratio 1:10 of plasmid/insert was chosen:

Ligase buffer 2x 2µL
Vector 1.2µL
Insert 5µL
Ligase 1µL
Purified water 10.8µL

Competent cells transformation was processed following the transformation protocol.

NB: because of the lack of a right 42°C water bath, the heatshock step was made at 37°C for 40 sec.

2 different plates were made: one at [Cm] = 25 g/L and the other at [Cm] = 12.5 g/L

The gel is not that clear: pSB1C3 without the insert is to high on the gel, but our transformant is a the good weight length : 3k3 bp. We will need to investigate further in order to be sure that we really have pSB1C3 + VhCqsA.

6 Clones, 2 from CM25 (25-1 and 25-2) and 4 from Cm 12.5 (12-1, 12-2, 12-3 and 12-4 ) that appear on Cm25 and Cm12.5 were grow on plate and on liquid media (5mL) LB + Cm 25 in order to miniprep them with the Miniprep protocol

Digestions were made on the plasmids obtained with the digestion protocol.

We identified a insert on our native pSB1C3, of around 600 bp, matching with the previous gels that we had. Also, 3 clone are ok: Clone1, 25-1 and 25-2. All of them have the wanted digestion: 2000 bp + 1300 bp. We will further investigate using PvuII, cutting twice, once on the insert, once on the vector.

In order to confirm the insert, two more digestion were made, with PvuII (which cut once on pSB1C3, and not on the insert) and SacI (which cut once on the insert, and once on pSB1C3) following the digestion protocol

The 3 plasmids were cut three time, as it was expected for the one containing CqsA sequence. Those 3 clone are validated, and have been stored on glycerol 25%. Their name will become: CqsA Anna (25-1), CqsA Maxant (C1) and CqsA Teo (25-2 )

Cloning of Vc_CqsA

The gel extraction from the 08/10 was digested using EcoRI and SpeI with the digestion protocol. The quantities for QSP 100 were:

  • 2.5µL of EcoRI
  • 2.5µL of SpeI
  • 30µL of DNA template
  • 10µL of Cutsmart
  • 55µL of water mQ

No heat inactivation were made. Instead, a PCR puriffication was processed using Sigma GenEtutTM PCR Clean Up kit. Afterward, a DNA quantification gel were made containing:

  • 2µL of ladder 1kb
  • 1µL of loading dye + 5µL of digested DNA
  • 1µL of loading dye + 5µL of digested pSB1C3
First, it is clear that the gel extraction worked fine. Second, it seems that the insert is twice the concentration of the digested plasmid.

Consequently, the ligation was carried out and lasted 50min. The followig quantities were used :

  • 2µL of ligase buffer
  • 1µL of ligase
  • 10.8µL of water mQ
  • 1.4µL of pSB1C3 digested E/S
  • 4.8µL of insert

Finally, Top 10 cells were transformed using the transformation (RbCl-method) protocol. Three aliquots were used:

  • 100µL of competent celles + 10µL of DNA: plate A
  • 100µL of competent cells + 5 µLof TE buffer + 5µL of DNA: plate B
  • 100µL of competent cells + 9µL of TE buffer + 1µL of pSB1C3 non digested (positive control): plate C

After O/N incubation, the followging observation were made on the Petri dishes:

  • Plate A: 12 transformants
  • Plate B: 19 transformants
  • Plate C: cell mat (even with a dilution x10)

Hence, eight transformants from each plate were put on liquid LB-Cm medium for subsequent mini prep. They were named A 1 to 8 and B 1 to 8. The transformants named were minipreped. The resulting DNA solutions were stored at -20°C.

After a quick analysis gel of all the miniprep, without any digestion (data not shown), 6 transformants seems to have the right plasmid lenght: A3, A4, A5, B3, B5, B6. Thus, these transformants were digested with PvuII and E/P to assess the right insertion of Vc CqsA.
Premix for PvuII (for 8 digestions):

  • 2 µL of pSB1C3 E/S + 8 µL of PvuII premix
  • 2 µL of A3 miniprep + 8 µL of PvuII premix
  • 2 µL of A4 miniprep + 8 µL of PvuII premix
  • 2 µL of A5 miniprep + 8 µL of PvuII premix
  • 2 µL of B3 miniprep + 8 µL of PvuII premix
  • 2 µL of B5 miniprep + 8 µL of PvuII premix
  • 2 µL of B6 miniprep + 8 µL of PvuII premix

Each tube was put in a water bath at 37°C for 1h. The resulting digestion were put on a 1% agarose gel as follows:

All of the transformants have the same profil and have both of the expected bands. Finally, the 6 transformants are 6 right clones Vc CqsA.

Clones A4 and A5 were named Vc CqsA 1 and Vc CqsA 2 and put in liquid culture to be stored at -80°C the next day.

Solid Bioluminescence assay

Four E. coli precultures were made in LB-Cm (5 mL):

  • Vh CqsA Max MG1655
  • Vh CqsA Anna MG1655
  • Vh CqsA Teo MG1655
  • pSB1C3 empty MG1655

Each tubes were complemented with 208 µL of a sterile glucose solution to reach a final concentration of 10 g/L. The tubes were put at 37°C O/N. In addition, two V. harveyi precultures were made:

  • LB: Vh WT (5 mL)
  • LB: JMH626 (10 mL)

They were put at 30°C O/N. OD of the O/N precultures were taken at 8.06 am. Therefore, fresh LB-Cm-Glc flasks of 10 mL were inoculated to reach OD = 0.1. (final glucose concentration in the medium: 10 g/L). Time of inoculation: 8.32 am. As OD = 0.3 had already been passed at 10.40 am, IPTG induction was made immediatly (final concentration : 0.5 mM). At 1 pm, the cultures were at the end of their exponential phase. Thus, the supernatants could be retrieved following the next steps:

  • pipeting of 10 mL of each of the cultures in (x5) eppendorf tube of 2 mL
  • centrifugation max speed, 5 min
  • pooling of each resulting supernatants
  • filtration through a 0.2 µm filter
  • storage at -20°C

OD of the V. harveyi O/N precultures were checked again at 11.10 am: JMH626: 4.15, Vh WT:3.11

(x5) LB flasks of 10 mL were inoculated with JMH626 to reach OD = 0.1. In addition, (x1) LB flask of 10 mL was inoculated with Vh WT, also at OD = 0.1. Time of inoculation: 11.28 am. When the OD was around 0.7, each of the 10 mL cultures were centrifugated at 4500 rpm for 6 min. The resulting supernatants were discarded while the pellets were resuspended with 5 mL of fresh LB medium and 5 mL of SN (one flask = one SN). Then, the cultures were put at 30°C. The whole process of resuspension was over at 3.30 pm.

In addition, 80 µL of the resuspended JMH cultures were dropped-off on a Petri dish and put at 30°C O/N. Acting as a landmark of bioluminescence, 80 µL of a Vh WT culture were also put on the Petri dish. For liquid cultures, the positive control showed bright bioluminescence as expected. Meawhile, all the cultures with the clones SN stayed dark. Regular checkings didn't bring more information. The Petri dish incubated O/N at 30°C was observed.

Test plan of the Petri dish (The Vh WT drop is the bioluminescence landmark)
The positive control, JMH626 culture supplemented with Vh WT SN is as bright as the Vh WT drop. Thus, SN from Vh WT can restore bioluminesence to its normal state, as far as human eyes can tell. The negative control shows faint bioluminscence, corresponding either to a basal expression of quorum sensing, or an activation of the quorum sensing by the HA1 ad AI-2 molecules produced naturally by E. coli. The second option seems quite unlikeky as LuxN and LuxQ (receptors of HA1 and AI-2 respectively) are knocked out in the JMH626 genome. SN of clones A and T show a bioluminescence as strong as the negative control, no real activity of C8-CAI-1 can be observed. SN of clone M seems to restore bioluminescence to normal state, as for the positive control.

Additional experiments need to be performed to conclud on these bioluminescence essays. Particularly, bioluminescence of JMH626 without any supplementation has to be tested.

Digestion, ligation and transformation of Vh1-pBR322

Analytical gel: digestion control (07/17)
  • 06/20: pBR322 (amplified the 06/19) was digested with EcoRI-HF and PstI-HF. Gel migration and gel extraction were performed to keep the vector.
  • 07/17: Vh1 part (PCR from IDT part) was digested with EcoRI-HF and PstI-HF, and purified with PCR purif kit.
  • 07/20: Ligation with T4 DNA Ligase and associated buffer from New England Biolabs was performed with pBR322 and Vh1 both digested with EcoRI-HF and PstI-HF, and the ligation mix was transformed into E. coli Stellar competent cells.
  • 07/21: 6 transformants were observed.
  • 07/24: The 6 transformants were grown on liquid culture for plasmid extraction.
  • 07/25:Plasmids were extracted with miniprep kit. Analytical digestion was performed with EcoRI/PstI and PvuII/XhoI. Three transformants had the expected digestion profile and were stored for Vh1-Vh2-pBR322 cloning.

Digestion, ligation and transformation of Vh2-pBR322

Analytical gel before ligation (07/18)
  • 06/20: pBR322 (amplified the 06/19) was digested with EcoRI-HF and PstI-HF. Gel migration and gel extraction were performed to keep the vector.
  • 17/07: Vh2 part (PCR from IDT part) was digested with EcoRI-HF and PstI-HF, and purified with PCR purif kit.
  • 18/07: Ligation with T4 DNA Ligase and associated buffer from New England Biolabs was performed with pBR322 and Vh1 both digested with EcoRI-HF and PstI-HF, and the ligation mix was transformed into E. coli Stellar competent cells.
  • 07/26: Observed transformants were grown on liquid culture.
  • 07/26: Plasmids were extracted with miniprep kit. Analytical digestion was performed with EcoRI/PstI and PvuII/XhoI. All the transformants had the expected digestion profile and were stored for Vh1-Vh2-pBR322 cloning.

Digestion, ligation and transformation of Vh3-pSB1C3

Analytical gel before ligation (07/12)
  • 08/12: Vh3 part (PCR from IDT), previously digested EcoRI-HF and PstI-HF, was digested with EcoRI-HF and SpeI-HF and purified with PCR purif kit. pSB1C3-Ter (BBa_1006) was digested with EcoRI-HF and SpeI-HF and gel extracted. Ligation with T4 DNA Ligase and associated buffer from New England Biolabs and transformation were performed. E. coli Top10 competent cells previously prepared were used.
  • 08/14: Eight transformants were observed on the transformation plates and six of them were grown on liquid culture.
  • 08/15: Plasmid extraction with miniprep kit and analytical digestion of the 6 transformants with EcoRI/PstI and ApaI/NcoI. The two transformants with the expected digestion profile were stored for cloning into conjugative plasmid and for diacetyl production in E. coli.

Digestion, ligation and transformation of Vh1-Vh2-pBR322

  • 08/07: Vh1-pBR322 and Vh2-pBR322 were digested with EcoRI-HF/XhoI (EX) to try the ligation Vh1 part into Vh2-pBR322. Gel migration and gel extraction were performed with these digestion mix.
  • 08/17: Ligations was performed: Vh1 EX (insert) with Vh2-pBR322 EX (vector). T4 DNA Ligase and associated buffer from New England Biolabs were used. E. coli Top10 competent cells previously prepared were used for transformation of ligation mix.
  • 08/18: Transformants were observed on the transformation plate. They were grown on liquid culture for plasmid extraction and analytical digestion.
  • 08/19: Plasmid extraction was performed with miniprep kit for all the transformants. The plasmids were then digested with EcoRI-HF/PstI-HF and BamHI/XhoI. All the transformants had the expected profile. They were stored for a Vh1-Vh2-Vh3 cloning.

Digestion, ligation and transformation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5

Analytical gel before ligation (13/09)
pBBR1MCS-4 (1) and pBBR1MCS-5 (1) were used for ligation
  • 08/15: Plasmid extraction was performed using miniprep kit. Preparative digestion of the conjugative plasmids pBBR1MCS-4 and pBBR1MCS-5 with EcoRI-HF and SpeI-HF (ES) from New England Biolabs was performed.
  • 09/14: Ligation with T4 DNA ligase and associated buffer from New England Biolabs was performed with pBBR1MCS-4 ES and pBBR1MCS-5 ES (vector) with RFP ES (BBa_J04450, insert). The RFP was initially digested by the Pichia pastoris module for its own constructions. The ligation mix was transformed into E. coli Top10 competent cells previously prepared.
  • 09/19: Red transformants were observed on the transformation plates for ligation RFP + pBBR1MCS-4 and RFP + pBBR1MCS-5. They were grown on liquid culture, and the two strains (one for pBBR1MCS-4 and one for pBBR1MCS-5) which seem to have the higher RFP activity were used for conjugation.

Conjugation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5 in Vibrio harveyi JMH626

  • 09/21: E. coli pBBR1MCS-4 AmpR, E. coli pBBR1MCS-5 GmR, V. harveyi JMH626 CmR KanR and E. coli pRK2073 SpcR helper were grown overnight on 5 mL liquid culture with appropriate antibiotics. E. coli were grown at 37°C and V. harveyi at 30°C.
  • 09/22: Centrifugation and resuspension in LB was performed for each liquid culture. The following conjugation mix were prepared:
    • Conjugation AmpR: E. coli pBBR1MCS-4 - RFP + V. harveyi JMH626 + E. coli pRK2073
    • Conjugation GmR: E. coli pBBR1MCS-5 - RFP + V. harveyi JMH626 + E. coli pRK2073
    • Negative control AmpR: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
    • Negative control GmR: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
    Each mix was deposed on a membrane upon a LB plate. The plates were incubated overnight at 30°C.
  • 09/23: The membranes were resuspended into water and the suspensions were spread on a new LB plate with double antibiotic selection: Amp and Cmp for “Conjugation AmpR” and “Negative control AmpR”, Gm and Cmp for “Conjugation GmR” and “Negative control GmR”. The plates were incubated overnight at 30°C.
  • 09/24: An uncountable quantity of colonies was observed on the two conjugation plates. No colonies were observed on the negative controls, which proves that conjugation worked. The cells were not red yet, and the contact inhibition on the plate (because of the too high quantity of colonies) may alter it aspects. Thus, the conjugation cells were replated on another plate with the same double antibiotic selection.
  • 09/25: The replated Vibrio harveyi cells showed a red fluorescent activity compared to a plate with V. harveyi JMH626 without conjugation.
Plates obtained from conjugation
Left: negative control / center: conjugation / right: replated conjugation colonies
Above: with pBBR1MCS-4 / Below: with pBBR1MCS-5
Comparison of JMH626 from conjugation (RFP-pBBR1MCS-4) and JMH626 (no conjugation)
Comparison of JMH626 from conjugation (RFP-pBBR1MCS-5) and JMH626 (no conjugation)
  • 09/26: V. harveyi JMH626 with pBBR1MCS-4, V. harveyi JMH626 with pBBR1MCS-5, V. harveyi JMH626 without plasmid (control) were grown on liquid culture overnight with antibiotics and LB at 30°C.

PCR of the plasmid pPICZαA

PCRs were processed on the pPICZαA coming from miniprep (cf section 1): PCR protocol

The aim of this PCR was to obtain a linear plasmid with 2 specific restriction sites at its extremity (BamHI and KpnI).

Once the PCR was over a gel migration (at 100 V, during 30 min) was performed: Gel migration protocol

pPICZα was not pure but we decided to do a gel extraction of the band of interest after the digestion. So PCR products went through a PCR purification.

Nanodrop of the PCR product: [pPICZαA] = 260,5 ng/µL

Once we knew the DNA concentration we decided to do the preparative digestion (BamHI-KpnI-HF): Digestion protocol

A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol

The band of interest at 1800 pb was purified via the Gel extraction protocol.

PCR of the insert Odr10-cOT2

PCRs was processed on the part pGAP-Odr10-pFUS1-cOT2 received from IDT: PCR protocol

Once the PCR was over a gel migration (at 100 V, during 30 min) was performed: Gel migration protocol

Odr10-cOT2 was not pure but we decided to do a gel extraction of the band of interest after the digestion. So PCR products went through a PCR purification.

Nanodrop of the PCR product: [Odr10-cOT2] = 277,3 ng/µL

Once we knew the DNA concentration we decided to do the preparative digestion (BamHI and KpnI-HF): Digestion protocol

A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol

The band of interest at 2800 pb was purified via the Gel extraction protocol.

PCR of the inserts pGAP-AMP

PCRs were processed on the parts pGAP-leucrocine, pGAP-cOT2, pGAP-DNY15 received from IDT: PCR protocol

pGAP-AMP = pGAP-leucrocine, pGAP-cOT2, pGAP-DNY15

A gel migration was performed, in order to check the PCRs. Gel migration protocol

The PCR products of the pGAP-AMPs were pure so a PCR purification was made after the different PCR had been pooled.

Nanodrop of the PCR products:

  • [pGAP-cOT2] = 185 ng/µl
  • [pGAP-leucrocine] = 185 ng/µl
  • [pGAP-cOT2] = 277 ng/µl

The 3 pGAP-AMP were digested with KpnI-HF and BamHI : Digestion protocol

Enzymes were removed while using a PCR purification kit.

Quantification of DNA concentration for ligation

pPICZαA and Odr10-cOT2 coming from a gel extraction were quantify thanks to a gel because the nanodrop couldn’t give precise quantity.

Their concentration was estimated at 30 ng/µl while pGAP-AMP went through the nanodrop after the PCR purification.

  • [pPICZα] = 30 ng/µl
  • [Odr10-cOT2] = 30 ng/µl
  • [pGAP-AMP] = 70 ng/µl

The Ligation Protocol with T4 DNA Ligase (M0202) has been followed.

Finally, competent cells transformation of E. coli DH5α was processed following the protocol: Transformation protocol. (NB: competent cells were plated on LB medium [zeo] = 25 g/L.)

Several clones were put in liquid culture of 5 ml of LB with [zeo] = 25 g/l.

Restriction map of transformants

Plasmids extraction was performed on the previous culture of 5 mL E. coli transformants grown on LB liquid media with zeocin : Miniprep protocol

Digestion were then processed as follows: Digestion protocol

We do have:

  • 2 clones containing pGAP-DNY15
  • 3 clones containing pGAP-cOT2
  • 4 clones containing pGAP-leucrocine
  • 2 clones containing Odr10-cOT2

Sequencing of one clone of each pGAP-AMP

Result of the sequencing of the Clone n°1 of pGAP-DNY15, the clone n°4 of pGAP-leucro and the clone n°5 of pGAP-Odr10-cOT2.

There is no mutation in the αfactor-AMP sequences so these clones were used to clone AMP in pSB1C3 for iGEM registry.

Clonage of antimicrobial peptides in pSB1C3 iGEM parts

We have 3 AMP genes to clone in pSB1C3.

  • Odr10-cOT2 is digested to extract the pPICαA containing pFUS1 and cOT2.
  • pGAP-leucro is digested to extract the pPICZαA containing pGAP and leucro.
  • pGAP-DNY15 is digested to extract the DNY15 gene.

Digestions were processed as follows (SpeI-HF and EcoRI-HF): Digestion protocol

A gel migration (at 100 V, during 30 min) was performed with all digestions mix, in order to separate the DNA fragments: Gel migration protocol

The Gel extraction protocol has been followed.

  • The 4 400 pb band of Odr10-cOT2 was purified: pPICZα-pFUS1
  • The 400 pb band of Odr10-cOT2 was purified: cOT2
  • The 2 400 pb band of pGAP-leucro was purified: pPICZα-pGAP
  • The 300 pb band of pGAP-leucro was purified: leucro
  • The 300 pb band of pGAP-DNY15 was purified: DNY15

Ligation & transformation of DNY15; cOT2 and leucrocine in pSB1C3 (Ligation Protocol with T4 DNA Ligase (M0202))

Finally, competent cells transformation in E. coli DH5α was processed following the protocol: Transformation protocol. NB: competent cells were plated on LB medium [cm] = 25 g/L. 6 clones of each plate were grown into LB+Chloramphenicol (5 ml each tube).

Plasmids extraction was performed on the previous culture of 5 mL E. coli transformants grown on LB liquid media with chloramphenicol.

Miniprep was then processed as follows: Miniprep protocol

Digestions were then processed as follows: Digestion protocol

A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol. All clones with good gel migration profile were kept.

Integration of genic construction in P. pastoris genome

To integrate a genic construction in P. pastoris genome, the first step is to linearize the plasmid at the localization of the integration (for instance in our case we linearize in the GAP promotor to have an integration in this promotor).

Each genic construction was digested as follows: Digestion protocol

  • pGAP-DNY15
  • pGAP-leucro
  • pGAP-cOT2
  • Odr10-cOT2
  • pPICZα without insert was used as a negative control.

Once digested, each construction is electropored in P. pastoris following the Electroporation protocol.

Yeast after transformation were plated on YPD + [zeo] = 250 g/l. Every transformants were then replated on YPD + [zeo] = 1000 g/l to select clones with a higher rate of genomic integration.

pGAP-DNY15 clones on [zeo] = 1000 g/l

The PCR colony protocol was used to verify that every clone had the insert.

pGAP-DNY15: A gel migration to amplify the pGAP-DNY15 gene integrated in pichia genome (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol

Odr10-cOT2: A gel migration to amplify the Odr1 0-cOT2 gene integrated in pichia genome (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol

RT-PCR experiment

The P. pastoris strains containing pGAP-DNY15 or the empty plasmid were grown in 50mL YPD 40g/L of glucose for 4 days at 30°C in an agitating incubator. RNAs of both were extracted and a RT-PCR experiment has been done.

AMP production and cytotoxicity tests

D-NY15 production was performed with the P. pastoris clone E obtained previously. Two cultures were carried out: one for D-NY15 E and the other for the negative control (P. pastoris transformed with pPICZα without insert). Each clone was inoculated in 50 mL YPD 40 g/L glucose and grown for 4 days at 30 °C in an agitating incubator. 15mL of each supernatant culture were stored at 4°C while 35mL were freeze-dried and then resuspended in 3.5mL of water.

Cytotoxicity test on plate were made using the disc diffusion technique. 200µL of V. harveyi WT (OD600 around 0.5) 100 times diluted were spread on LB agar. Discs were soaked with supernatant, placed on the petri dish and incubated overnight at 30°C.

The same experiment was made again spreading 200µL of V. harveyi WT no diluted:

Protocols

PCR

Introduction

We used the Thermo Scientific Phusion High-Fidelity DNA Polymerase. Amplification of templates with high GC content, high secondary structure, low template concentrations or long amplicons may require further optimization.

Materials

  • PCR thermocycler
  • PCR tubes
  • nuclease-free water
  • dNTP
  • Phusion HF Buffer (X5) or GC Buffer
  • Primers (both forward and reverse)
  • Template DNA
  • Phusion polymerase

Procedure

All components should be mixed and centrifuged prior to use. It is important to add Phusion DNA Polymerase last in order to prevent any primer degradation caused by the 3´→ 5´ exonuclease activity.

Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors.

Use of high quality, purified DNA templates greatly enhances the success of PCR.

  1. We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
    Component 50 μL final concentration
    Nuclease-free water qs 50 μL
    Buffer Phusion HF (5X) 10μL 1X
    10 mM dNTPs 1 μL 200 μM
    10 μM Forward primer 2.5 μL 0.5 μM
    10 μM Reverse Primer 2.5 μL 0.5 μM
    DNA template (10 ng/μL) 1 μL 10ng
    Phusion DNA Polymerase 0.5 μL 1.0 U/0.5 μL of reaction
    Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary
  2. Transfer PCR tubes from ice to a PCR machine with the block preheated to 98°C and begin thermocycling:
    Step Temperature Time
    Initial denaturation 98°C 45 sec
    30 cycles 98°C 15 sec
    55°C 30 sec
    72°C 30 sec/kb
    Final extension 72°C 5 min
    Hold 4°C hold
    Parts Length Time of extension
    pGAP-cOT2 / pGAP-DNY15 / pGAP-Leucro / YFP / DsRed 1 kb 30 sec
    harveyi 1 / 2 / 3 / Vc and Vh 2 kb 60 sec
    Odr10-cOT2 3 kb 90 sec
  3. Then purify the products thanks to PCR purification kit

Gel extraction

Procedure

Please, before doing your preparative gel, use one sample to make an analityc one !

  1. Equilibrate a water bath or heat block to 50°C.
  2. Excise a minimal area of gel containing the DNA fragment of interest.
    • Crucial: To protect the UV box, it is a good idea to place the gel on a glass plate if available.
    • Try to get as little excess gel around the band as possible.
  3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.
  4. Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:
    Gel Tube Buffer L3 Volume
    ≤2% agarose 1.7 mL polypropylene 3:1 (i.e., 1.2 mL Buffer L3: 400 mg gel piece)
    >2% agarose 5 mL polypropylene 6:1 (i.e., 2.4 mL Buffer L3: 400 mg gel piece)
  5. Place the tube with the gel slice and Buffer L3 into a 50°C water bath or heat block. Incubate the tube at 50°C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution.
    • Note: High concentration gels (>2% agarose) or large gel slices may take longer than 10 minutes to dissolve.
  6. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes.
    • Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
  7. Before Starting: Add ethanol to the Wash Buffer (W1) according to the label on the bottle.
  8. Purifying DNA Using a Centrifuge
  9. Load. Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel.
    • Note: The column reservoir capacity is 850 μL.
  10. Bind. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
  11. Wash. Add 500 μL Wash Buffer (W1) containing ethanol to the column.
  12. Remove Buffer. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
  13. Remove Ethanol. Centrifuge the column at maximum speed for 1–2 minutes. Discard the flow-through.
  14. Elute. Place the column into a Recovery Tube. Add 30 μL Elution Buffer (E5) to the center of the column. Incubate the tube for 1 minute at room temperature.
  15. Collect. Centrifuge the tube at >12,000 × g for 1 minute.
  16. Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at −20°C for long-term storage.

Gel migration

Introduction

This protocol is the classical one used for electrophoresis. - You can adapt the concentration of agar according to the length of your fragment 1% agar if the DNA fragments are big 2% agar if the DNA fragments are small (the bigger fragment are sticked together) - Adapt the volume of the gel 15 to 30 mL for small gels and 150 to 200 mL for big gels

Procedure

  1. Thoroughly rinse gel housing and well-comb with dH2O.
  2. Place gel mold perpendicular to flow direction, ensuring proper sealing of rubber gaskets.
  3. Add the calculated amounts of 0.5xTBE and agarose to a fresh Erlenmeyer flask.
  4. Heat in microwave until mixture can be dissolved.
    • CRITICAL: Do not let the mixture boil over and out of the flask. Typical heating time for 50mL in a 2.45GHz microwave oven at full power is 30s. USE HEAT GLOVES
  5. Gently swirl until well mixed and gently swirling periodically until ~55°C.
  6. Gently pour molten agarose gel into housing, avoiding air bubbles.
  7. Place desired well comb in desired position.
  8. Once gelled, carefully remove well comb in a uniform fashion.
  9. Remove gel mold and place in parallel direction to flow
    • CRITICAL: the deposit line has to be at the anode (negative pole)
  10. Fill gel box with 0.5 xTAE until the gel is well covered.
  11. Place the ladder on the gel, the native and digested plasmid (write down the gel map)
    • TIP: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip.
  12. Run the electrophoresis for 20-30min at 100V until the dye line is approximately 80% of the way down the gel
  13. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
  14. Place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, r
  15. Place the gel into a container filled with water and destain for 5 mins.
  16. Reaveal under UV lamp, visualize your DNA fragments

Miniprep

Introduction

This protocol was taken from the ThermoScientific GeneJET Plasmid Miniprep Kit. Safety: Both the Lysis Solution and the Neutralization Solution contain irritants. Wear gloves when handling these solutions.

Procedure

  • Note: All steps should be carried out at room temperature. All centrifugations should be carried out in a microcentrifuge at ≥ 12 000 x g (10 000-14 000 rpm, depending on the rotor type).
  • Be sure that the concentrated solutions have been diluted with the appropriated buffer
  1. Pick a single colony from a freshly streaked selective plate to inoculate 5mL of LB medium supplemented with the appropriate selection antibiotic.
  2. Incubate for 12-16 hours at 37°C while shaking at 200-250 rpm
  3. Centrifugate the bacterial culture, >12 000 g in a microcentrifuge for 2 minutes at room temperature. Repeat until there is no more media.
  4. Add to the pelleted cells:
    • 250 μL of Resuspension Solution and vortex
    • 250 μL of Lysis Solution and invert the tube 4-6 times. WAIT 2 min
    • 350 μL of Neutralization Solution and invert the tube 4-6 times.
    • Lysis buffer must be neutralized before 5 minutes
  5. Centrifuge 5 minutes.
  6. Transfer the supernatant to the Thermo Scientific GeneJET Spin Column. Centrifuge 1 minute
  7. Add 500 μL of Wash Solution and centrifuge for 60 s and discard the flow-through
  8. Repeat step 5.
  9. Centrifuge empty column for 1 minute.
  10. Dry for 5 minutes
  11. Transfer the column into a new tube.
  12. Add 30 μL of Elution Buffer to the column and incubate 2 minutes.
  13. Centrifuge 2 minutes.
  14. Collect the flow-through.