Team:NU Kazakhstan/Notebook

From 2014.igem.org

(Difference between revisions)
Line 124: Line 124:
<p>See agarose gel picture for the results.</p>
<p>See agarose gel picture for the results.</p>
<img src="https://2014.igem.org/File:June_19.jpg" width="100" height="200">
<img src="https://2014.igem.org/File:June_19.jpg" width="100" height="200">
 +
<p>June 20: We did pcr of backbones and rfp part again, but using 3 best annealing temperatures. We have also used concentration of primers for the backbone twice as low as last time, and the bands are very clear. See the picture</p>
 +
<img src="https://2014.igem.org/File:June_20.jpg" width="100" height="200">
 +
<p>June 21-22: We did transformation of e.coli with 3 part+1 control. PelB and HlyA worked well, but LigA, which was planned to be used as 75kDa protein did not work. I found out, that it requires the special KRX strain of e.coli, which we do not have. So, I took another part (Salty_VrgP) from the kit which 79kDa protein and did the transformation; also I did transformation with NahR gene, which gives 34kDa protein, we will use it in constructing "rehearsal" part of the "competence project". In addition, we did miniprep of plasmids, that we've got from Madrid and of PelB and HlyA.</p>
 +
<p>June-23: we did the competent dh5a cells. The transformation was conducted The cells are competent.</p>
 +
<p> July 19: We decided to check expression of VHamylase in E. coli from Madrid.</p>
 +
<ol>The whole protocol of what we did:
 +
<li>Inoculate a single colony of the bacteria from plate into 5ml LB with 100ng/ul Ampicillin, 33ng/ul, chloramphenicol, and 0.5mM IPTG. Shake for 12 hours at 150 rpm at 30degC</li>
 +
<li>Centrifuge the overnight colonies at 4000g for 40 min</li>
 +
<li>Take 4ml of the supernatant and mix with 800ul of 25% Trichloroacetic acid (TCA). Leave the samples at -20degC overnight</li>
 +
<li>Centrifuge the samples for 1hour at 14000g at +4degC, then wash twice with acetone and let dry the pellets for 2 hours at room temperature</li>
 +
<li>Resuspend the pellets in 100ul sample buffer (Laemmli+b-mercaptoethanol) and boil at 95C for 10min</li>
 +
<li>Run the samples on SDS PAGE, stain with Coomasie blue and then destain for 24 hours</li>
 +
<li>We observed the bands of size 41kDa. See the picture for the results, but it is not seen clearly on the picture, probably, because of low concentration of the protein.
 +
Note: Size of 41kDa indicates the fact that secreted protein was not dimerized because dimerized protein was expected to have the size of 84 kDa</li></ol>
 +
<img src="https://2014.igem.org/File:July_19.jpg" width="100" height="200">
 +
<p>July 19: We did restriction digest of miniprep product (GFP in plasmid backbone) using EcoRI and SpeI:
 +
<p>1. Digestion with SpeI: DNA - 2ul (500ng), 10xTango buffer - 2ul, Water - 15.5ul, SpeI (Fermentas) - 0.5ul. We incubated the reaction at 37C for 1.5 hour</p>
 +
<p>2. Then we added 2.5ul of EcoRI buffer and 0.5ul EcoRI (Fermentas) to the initial reaction.</p>
 +
<p>3. We ran the reaction mixture on the gel and observed that the digestion worked (see the picture, third well): we have got band for the plasmid backbone (2070bp) and for GFP (~700bp)</p>
 +
<img src="https://2014.igem.org/File:July_19_restriction_digest.jpg" width="100" height="200">
 +
<p>July 21: we decided to do the double digest of plasmid with EcoRI & PstI.</p>
 +
<p>Total reaction mixture - 20ul: DNA miniprep product (700ng) - 2.0ul,10xBuffer SH - 2ul,EcoRI (Invitrogen) - 1ul, PstI (Sigma) - 1ul. We incubated the reaction at 37C for 1hour, then inactivated the enzymes at 65C for 15 min.
 +
The double digestion worked: we were able to see two distinct bands (for plasmid backbobe and for GFP). See the image.</p>
 +
<img src="https://2014.igem.org/File:July_21.jpg" width="100" height="200">
 +
 +
<img src="https://2014.igem.org/File:July_27_CPEC.jpg" width="100" height="200">
 +
<img src="https://2014.igem.org/File:July_27.jpg" width="100" height="200">
 +
<img src="https://2014.igem.org/File:July_29_CPEC.jpg" width="100" height="200">
  </td>         
  </td>         
</td>
</td>

Revision as of 10:10, 13 August 2014






May 12

Congratulations with the First Working Day!


    May 13:
  • Camelid's antibody p53 sequence was found
  • Exploring camelid antibodies:

    • 1) Nanobodies: Natural Single-Domain Antibodies by Serge Muyldermans, March 2013

    2) Nanobody-based products as research and diagnostic tools by Thomas De Meyer et al, 2013

May 17: We recieved the reply from Dr. Fernandez lab who are ready to send the plasmid containing the zipper and HlyA sequence. Special thanks to Dr. Shipley who wrote the letter.

May 19: We had discussed the three topics, the nanobody (nb) project's protocol is ready, antimicrobial peptides (amp) and phi29 are under the process. We distributed the people from nb group to support othre projects. The new papers and nb presentation were uploaded on the google drive

June 4: We did DNA miniprep of E. coli cultures in LB medium, which was obtained from plates with transformed plates. Cells were transformed with the parts from the kit: BBa_K608002 and BBa_E1010. The tubes with miniprep products are at +4C

    Concentrations of DNA measured with Nanodrop:
  • Promoter: 221.3 ng/ul
  • RFP: 348.2 ng/ul
  • RFP from Arabinose plates: 230.2 ng/ul

June 13:

We did miniprep of DNA from sigle colony isolation (see June 11) and checked our DNA concentration on nanodrop: [BBa_I15016]=206.7ng/ul and [RFP control]=375.1ng/ul

June 16. Dear team! I am glad to inform you that we have received stab of E. coli with the plasmid containing the zipper and HlyA sequence, and a stab of E. coli with pVDL9.3 encoding hlyB and hlyD!!! A big thank to Dr. Fernandez Herrero and to Dr. Shipley!

June 19: We did pcr of Chloramphinecol plasmid backbones, and we used the following reagent mixture set up for 20ul:

  • Water-5ul
  • High fidelity master mix-10ul
  • Primer forward-1ul (we took from 50ul of 10uM stock)
  • Primer reverse-1ul (we took from 50ul of 10uM stock)
  • DNA template (0.2ng/ul)-3ul.
  • PCR temperature set-up:
  • Initial denaturation: 98C, 30s
  • Denaturation: 98C, 10s
  • Annealing: Temperature gradient from 57C to 71C, 30s
  • Extension: 72C, 41s
  • Final extension: 72C, 5min

    • See agarose gel picture for the results.

    June 20: We did pcr of backbones and rfp part again, but using 3 best annealing temperatures. We have also used concentration of primers for the backbone twice as low as last time, and the bands are very clear. See the picture

    June 21-22: We did transformation of e.coli with 3 part+1 control. PelB and HlyA worked well, but LigA, which was planned to be used as 75kDa protein did not work. I found out, that it requires the special KRX strain of e.coli, which we do not have. So, I took another part (Salty_VrgP) from the kit which 79kDa protein and did the transformation; also I did transformation with NahR gene, which gives 34kDa protein, we will use it in constructing "rehearsal" part of the "competence project". In addition, we did miniprep of plasmids, that we've got from Madrid and of PelB and HlyA.

    June-23: we did the competent dh5a cells. The transformation was conducted The cells are competent.

    July 19: We decided to check expression of VHamylase in E. coli from Madrid.

      The whole protocol of what we did:
    1. Inoculate a single colony of the bacteria from plate into 5ml LB with 100ng/ul Ampicillin, 33ng/ul, chloramphenicol, and 0.5mM IPTG. Shake for 12 hours at 150 rpm at 30degC
    2. Centrifuge the overnight colonies at 4000g for 40 min
    3. Take 4ml of the supernatant and mix with 800ul of 25% Trichloroacetic acid (TCA). Leave the samples at -20degC overnight
    4. Centrifuge the samples for 1hour at 14000g at +4degC, then wash twice with acetone and let dry the pellets for 2 hours at room temperature
    5. Resuspend the pellets in 100ul sample buffer (Laemmli+b-mercaptoethanol) and boil at 95C for 10min
    6. Run the samples on SDS PAGE, stain with Coomasie blue and then destain for 24 hours
    7. We observed the bands of size 41kDa. See the picture for the results, but it is not seen clearly on the picture, probably, because of low concentration of the protein. Note: Size of 41kDa indicates the fact that secreted protein was not dimerized because dimerized protein was expected to have the size of 84 kDa

    July 19: We did restriction digest of miniprep product (GFP in plasmid backbone) using EcoRI and SpeI:

    1. Digestion with SpeI: DNA - 2ul (500ng), 10xTango buffer - 2ul, Water - 15.5ul, SpeI (Fermentas) - 0.5ul. We incubated the reaction at 37C for 1.5 hour

    2. Then we added 2.5ul of EcoRI buffer and 0.5ul EcoRI (Fermentas) to the initial reaction.

    3. We ran the reaction mixture on the gel and observed that the digestion worked (see the picture, third well): we have got band for the plasmid backbone (2070bp) and for GFP (~700bp)

    July 21: we decided to do the double digest of plasmid with EcoRI & PstI.

    Total reaction mixture - 20ul: DNA miniprep product (700ng) - 2.0ul,10xBuffer SH - 2ul,EcoRI (Invitrogen) - 1ul, PstI (Sigma) - 1ul. We incubated the reaction at 37C for 1hour, then inactivated the enzymes at 65C for 15 min. The double digestion worked: we were able to see two distinct bands (for plasmid backbobe and for GFP). See the image.

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