Difference between revisions of "Team:Vilnius-Lithuania/Labjournal"

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09 07 – Decided what volume of protein should be used for western blot. <br />
 
09 07 – Decided what volume of protein should be used for western blot. <br />
 
09 10 – Visualized Western Blot results.</p>
 
09 10 – Visualized Western Blot results.</p>
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  </div>
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</div>
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</div>
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</div>
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</div>
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<!-- /MethodBox -->
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<!-- MethodBox -->
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                <div class="panel panel-default" style="position: relative">
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                    <div class="panel-heading">
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                        <a class="accordion-toggle collapsed" data-toggle="collapse" data-parent="#accordion" href="#10">
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                        <div class="panel-title">
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                        <h3 style="padding: 0px; margin: 0px;">System characterization</h3>
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<div class="row">
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  <div class="col-md-6">
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Parts:
 +
<ul style="list-style: none; padding: 0px; margin: 0px;">
 +
<li><strong>Cd21L26</strong> (BBa_K1773022)</li>
 +
<li><strong>Cd21L27</strong>(BBa_K1773023)</li>
 +
<li><strong>Cd21L28</strong>(BBa_K1773024)</li>
 +
</ul>
 +
  </div>
 +
  <div class="col-md-6">
 +
Written by: <strong>BB</strong><br />
 +
Performed by: <strong>BB and IO</strong>
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  </div>
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</div>
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                        </div>
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                        </a>
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                    </div>
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                    <div id="10" class="panel-collapse collapse">
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                        <div class="panel-body">
 +
<div class="row">
 +
  <div class="col-md-12">
 +
  <h3><strong>About</strong></h3>
 +
  <p>
 +
Cd21L26 - Cascade with pLux/cI right promoter and strong RBS <br />
 +
Cd21L27 - Cascade with pLux/cI right promoter and Medium RBS <br />
 +
Cd21L28 - Cascade with pLux/cI right promoter and weak RBS <br />
 +
</p>
 +
  </div>
 +
</div>
 +
 +
<div class="row">
 +
  <div class="col-md-12">
 +
  <table class="table">
 +
    <thead>
 +
      <tr>
 +
        <th>Date</th>
 +
        <th>Materials</th>
 +
        <th>Experiment</th>
 +
      </tr>
 +
    </thead>
 +
    <tbody>
 +
      <tr>
 +
        <td>09 04</td>
 +
        <td>
 +
SP1<br />
 +
SP3<br />
 +
pAcYc plasmid<br />
 +
DH5α<br />
 +
Karbenicilin</td>
 +
        <td>
 +
Restriction of pCDF vector, SP1 and SP3 genes with EcoRI and XbaI. <br />
 +
Ligating pieces afterwards, transforming new pCDF plasmid into bacteria. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 05</td>
 +
        <td>
 +
DH5α transformants<br />
 +
Bl21-DE3 transformants<br />
 +
pCola-Cas1-Cas3 plasmid<br />
 +
Chloramphenicol<br />
 +
Karbenicilin </td>
 +
        <td>
 +
Colony PCR. Inoculating good colonies.<br />
 +
Inoculating Bl21-DE3 transformants (BL21-DE3 with Cd21L26 + HCsy3; Cd21L27 + HCsy3; Cd21L28 + HCsy3) overnight. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 06</td>
 +
        <td>
 +
Bl21-DE3 transformants<br />
 +
pCola-Cas1-Cas3 plasmid<br />
 +
SP1<br />
 +
SP3<br />
 +
pCDF<br />
 +
DH5α bacteria<br />
 +
Chloramphenicol<br />
 +
Kanamycin<br />
 +
Streptomycin </td>
 +
        <td>
 +
Inoculating night culture of Bl21-DE3 transformants for 3 hours. <br />
 +
Preparing competent cells. Transforming pCola plasmid into BL21-DE3 transformants. <br />
 +
Repeat cloning of SP1 and SP3 into pCDF vector and transforming them into DH5α. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 07</td>
 +
        <td>
 +
DH5α transformants<br />
 +
Bl21-DE3 transformants<br />
 +
pCola-Cas1-Cas3 plasmid<br />
 +
Chloramphenicol<br />
 +
Karbenicilin <br />
 +
Kanamycin <br />
 +
Streptomycin </td>
 +
        <td>
 +
Colony PCR. Inoculating good SP1 and SP3 colonies overnight. 
 +
Repeating yesterdays transformation.  Inoculating night colonies again, in case of transformation failure. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 08</td>
 +
        <td>
 +
DH5α transformants<br />
 +
Bl21-DE3 transformants<br />
 +
pCola-Cas1-Cas3 plasmid<br />
 +
Chloramphenicol<br />
 +
Karbenicilin <br />
 +
Kanamycin </td>
 +
        <td>
 +
Extracting SP1 and SP3 plasmids. Repeating transformation. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 09</td>
 +
        <td>
 +
Bl21-DE3 bacteria<br />
 +
pCola-Cas1-Cas3 plasmid<br />
 +
Cd21L26 plasmid<br />
 +
Cd21L27 plasmid<br />
 +
Cd21L28 plasmid<br />
 +
Chloramphenicol<br />
 +
Kanamycin </td>
 +
        <td>
 +
Transformation of pCola and Cd21L26 into BL21-DE3 bacteria.<br />
 +
Transformation of pCola and Cd21L27 into BL21-DE3 bacteria.<br />
 +
Transformation of pCola and Cd21L28 into BL21-DE3 bacteria.</td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 10</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
Chloramphenicol<br />
 +
Kanamycin </td>
 +
        <td>Inoculating transformants overnight. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 11</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
pCDF-SP1 plasmid<br />
 +
pCDF-SP3 plasmid<br />
 +
Chloramphenicol<br />
 +
Kanamycin<br />
 +
Streptomycin</td>
 +
        <td>
 +
Measuring night culture optical dense. Equalizing optical dense of all bacteria.<br />
 +
Preparing cells for transformation. Transforming pCDF-SP1, pCDF-SP3 and pCDF plasmids into BL21-DE3 transformants. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 12</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
pCDF-SP1 plasmid<br />
 +
pCDF-SP3 plasmid<br />
 +
Chloramphenicol<br />
 +
Kanamycin<br />
 +
Streptomycin</td>
 +
        <td>
 +
Repeating transformation. Before inoculating bacteria on a Petri dish, it is diluted 100 and 10000 times.</td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 13</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
pCDF-SP1 plasmid<br />
 +
pCDF-SP3 plasmid<br />
 +
Chloramphenicol<br />
 +
Kanamycin<br />
 +
Streptomycin</td>
 +
        <td>
 +
Repeating transformation.<br />
 +
Before inoculating bacteria on a petri dish, it is diluted 100 and 1000 times. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 14</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
pCDF-SP1 plasmid<br />
 +
pCDF-SP3 plasmid<br />
 +
Chloramphenicol<br />
 +
Kanamycin<br />
 +
Streptomycin</td>
 +
        <td>
 +
Repeating transformation. <br />
 +
Before bacteria inoculation on a petri dish, it is diluted 100 and 10000 times.</td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 15</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
Chloramphenicol<br />
 +
Kanamycin</td>
 +
        <td>
 +
Inoculating new BL21-DE3 transformant cells. Growing overnight. </td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 16</td>
 +
        <td>
 +
BL21-DE3 transformants<br />
 +
pCDF-SP1 plasmid<br />
 +
pCDF-SP3 plasmid<br />
 +
Chloramphenicol<br />
 +
Kanamycin<br />
 +
Streptomycin</td>
 +
        <td>
 +
Repeating transformation. Before bacteria inoculation on a petri dish, it is diluted 100 and 10000 times.</td>
 +
      </tr>
 +
      <tr>
 +
        <td>09 17</td>
 +
        <td>
 +
BL21-DE3 transformants </td>
 +
        <td>
 +
Counting grown colonies. </td>
 +
      </tr>
 +
    </tbody>
 +
  </table>
 +
  </div>
 +
</div>
 +
 +
<div class="row">
 +
  <div class="col-md-12">
 +
  <h3><strong>Conclusions and Results:</strong></h3>
 +
  <p style="height: auto; margin: auto;">
 +
09 05 – Transformation successful. Colony PCR showed, that SP1 and SP3 fragments were cloned successfully. <br />
 +
09 06 – Mistake found. SP1 and SP3 were cloned into a wrong vector. <br />
 +
09 07 – Transformation was unsuccessful.<br />
 +
09 08 – Transformation unsuccessful. Trying to reduce antibiotic quantity. <br />
 +
09 09 – Transformation unsuccessful. PET-HCsy3 plasmid may have inhibited transformation.  <br />
 +
09 10 – Transformation successful.  <br />
 +
09 11 – BL21-DE3 transformed with Cd21L27+pCola did not grow.  <br />
 +
09 12 – Too much bacteria grew up.  <br />
 +
09 13 – Transformation unsuccessful. In some dishes still too many bacteria grown.  <br />
 +
09 14 – Transformation unsuccessful.  <br />
 +
09 15 – Transformation unsuccessful. Probably not enough pCDF-SP1 and pCDF-SP3 vector.  <br />
 +
09 17 - Transformation successful.</p>
  
 
   </div>
 
   </div>

Revision as of 09:45, 18 September 2015

About

pLac+cI.
P10 and P11 plasmids are used. pLac (P11) and repressor cI (P10) ligation.

Date Materials Experiment
06 30 P10
P11		 P10 and P11 transformation into DH5α.
07 01 Bacteria inoculation (4 minipreps).
07 09 P10
P11		 Plasmids’ restriction (3 ug each).
P10 with EcoRI and XBal (+FastAp).
P11 with EcoRI and SpeI.
Extraction from gel
L10 and L11 are produced.
Conc.: L11 – 2,4 ng/μL.
07 13 P10 Repeated restriction of P10 with EcoRI and XBal (+FastAp).
Conc.: L10 – 45,3 ng/μL. L10 and L11 ligation.
P14 is produced.
07 14 Bacteria with P14 Colony PCR of P14 bacteria. 1st and 2nd colonies are inoculated.
P14.2 Sequencing

Conclusions and Results:

07 14 – all of the colonies are successful, except third colony.

About

Cas3 with mutated restriction sites.
L7+L15. Concentration - 261 ng/μL

Date Materials Experiment
06 30 P13
P7		 P13 and P7 transformation into DH5α.
07 01 Transformants are inoculated (4 minipreps each).
07 02 Plasmid extraction. Concentrations:
P7 – 174,1 ng/μL and 166 ng/μL.
P13 - 216 ng/μL and 131, 5 ng/μL.
07 13 P13 Cas3 mutagenesis (with ST1/2, ST3/4, ST5/6 primers).
07 16 Mutated P13 Cas3 plasmids extraction.
07 21-23 Mutated P13 Restriction with EcoRI, XbaI and PstI.
07 27 Mutated P13 Cas3 mutagenesis.
07 29 P7 Restriction with EcoRI and SpeI. L7 is produced.
07 31 P13
08 02 Mutated P13 Mutated Cas3 restriction with EcoRI, XbaI and PstI.
08 05 Mutated P13 Cas3 mutagenesis.
08 07 Mutated P13 Mutated Cas3 restriction with PstI. Mutagenesis was successful.
L15 is produced.
08 11 L7
L15		 L7 and L15 ligation with pSB1C3 (EcoRI+XBal+FastAp).
P15.1 is produced.
P15.1 Sequencing.

Conclusions and Results:

07 21 – Mutagenesis was unsuccessful.
08 02 – successfully mutagenized EcoRI and XbaI sites.
08 07 – successful mutagenesis confirmed (XbaI and EcoRI sites are mutated).
08 07 – Successful mutagenesis (PstI, XbaI and EcoRI sites are mutated).

About

P26 – L7+L26 pLux/cI+SRBS
P27 – L7+L27 pLux/cI+MRBS
P28 – L7+L28 pLux/cI+WRBS
P29 – L7+L29 pLux/cI

Date Materials Experiment
07 27 ST9
ST10
ST11
ST12
ST15
ST16
ST17
ST18
ST19
ST20		 pLux/cI contruction from oligos – PCR reaction with ST9 and ST10/11/12 primers, then with
ST15 and ST16/17/18.
Parallel PCR with ST9 and ST19 and then with ST15 and ST20.
PCR products purification.
07 28 PCR products from oligos restriction with EcoRI and SpeI.
L26, L27, L28 and L29 are produced.
07 28 L7
L26
L27
L28
L29		 Ligation of:
L7 and L26, L7 and L27, L7 and L28, L7 and L29
and their transformation into DH5α.
07 29 -
- 08 05		 L7+L26 colony PCR with ST15 and VR.
L7+L27 colony PCR with ST15 and VR.
L7+L28 colony PCR with ST15 and VR.
1st and 2nd colonies are inoculated.
P26, P27, 28 and P29 are produced
P26, P27, 28 and P29 restriction with NspI.
08 10 P26, P27, 28 and P29 sequencing with VF2.

Conclusions and Results:

07 31 – all of the colonies are successful.
08 10 – all biobricks are sequenced succesfully.

About

cI repressor promoter (BBa_R1051) (P4) + screen plasmid intermediate (GFP gene) (BBa_I13504) (P5).

Date Materials Experiment
06 30 P14 P4 transformation into DH5α.
07 01 Bacteria inoculation (4 minipreps).
07 02 P4 Plasmid extraction. Concentrations:
P4 - 238,3 ng/μL and 224,3 ng/μL.
P4 restriction with EcoRI and XBal.
Extraction from gel.
L4 is produced (con. 16,2 ng/μL).
07 07 P5 P5 transformation into DH5α.
Bacteria inoculation.
07 08 P5 plasmid extraction (P5.1, P5.2, P5.3, P5.4, P5.5 are produced).
07 29 P5.1 and
P5.3		 Restriction with EcoRI and SpeI.
Extraction from gel.
L5.1A, L5.1B, L5.1C and
L5.3A, L5.3B, L5.3C are produced.
Conc.: L5.1A – 0,6 ng/μL, L5.1B – 0,6 ng/μL, L5.1C – 2,4 ng/μL,
L5.3A – 0,7 ng/μL, L5.3B - 3 ng/μL, L5.3C – 5,1 ng/μL.
Ligation of L4 and L5.1, L4 and L5.3.
P30 is produced.
08 01 P30 Sequencing.

Conclusions and Results:

07 09 – fragments were not the right ones.
07 29 – the right fragments are produced.

About

P35 - pLux/cI+SRBS(Strong RBS)+Cas3 (L26+L15.1)
P36 - pLux/cI+MRBS(Medium RBS)+Cas3 (L27+L15.1)
P37 - pLux/cI+WRBS(Weak RBS)+Cas3 (L28+L15.1)
P26 – L7+L26 (pLux/cI+SRBS)
P27 - pLux/cI+MRBS(Medium RBS)
P28 - pLux/cI+WRBS(Weak RBS)

Date Materials Experiment
07 28 P15 Mutated Cas3 with Preffix and Suffix restriction with EcoRI and XbaI.
L15 is produced.
07 28 P26
P27
P28		 PCR products from oligos restriction with EcoRI and SpeI.
L26, L27, L28 and L29 are produced.
07 28 L15
L26
L27
L28		 Ligation of: L15 and L26, L15 and L27, L15 and L28,
and their transformation into DH5α.
07 29 -
- 08 05		 L7+L26 colony PCR with ST15 and VR.
L7+L27 colony PCR with ST15 and VR
L7+L28 colony PCR with ST15 and VR
1st and 2nd colonies are inoculated.
P35, P36 and P37 are produced
P35, P36, P37 restriction with NspI.
08 10 P35, P36, P37 sequencing.

Conclusions and Results:

07 31 – all of the colonies are successful.
08 10 – all biobricks are sequenced succesfully.

About

Mutated Cascade protein.

Date Materials Experiment
06 30 Cd Cd transformation into DH5α.
07 01 Transformants are inoculated (4 minipreps each).
07 02 Plasmid extraction. Conc.: 329 ng/μL and 294 ng/μL.
07 14 Cd Cd mutagenesis (with ST13/14 primers).
07 16 - 17 Mutated plasmids extraction. Restriction with EcoRI.
07 19 Mutated Cd Restriction repeat.
07 21 Mutated Cd Repeated mutagenesis (with ST13/14).
07 27 Mutated Cd Mutated plasmids extraction. Mutated Cd restriction.
08 10 Mutated Cd
L7		 PCR with mutated Cd with ST26/27.
PCR product purification.
Conc.: 63 ng/μL.
Restriction with EcoRI and SpeI.
Mutated Cd ligation with L7.
Cd1 is produced.
Cd1 Sequencing.

Conclusions and Results:

07 16-17 – Mutagenesis was unsuccessful.
07 27 – Mutagenesis conformed as a successful.

About

26-Cd - Cascade with SRBS
27-Cd - Cascade with MRBS
28-Cd - Cascade with WRBS

Date Materials Experiment
08 15 Cd1 Mutated Cd with Preffix and Suffix restriction with EcoRI and XbaI. L35 is produced.
08 15 P26
P27
P28
 PCR products from oligos restriction with EcoRI and SpeI. L26, L27, L28 and L29 are produced.
08 15 L35
L26
L27
L28
 Ligation of: L35 and L26, L35 and L27, L35 and L28,
and their transformation into DH5α.
08 17 -
- 08 20		 L35+L26 colony PCR with ST15 and VR.
L35+L27 colony PCR with ST15 and VR
L35+L28 colony PCR with ST15 and VR
1st and 2nd colonies are inoculated.
26-Cd, 27-Cd and 28-Cd are produced.
26-Cd, 27-Cd and 28-Cd restriction with NspI.
08 21 26-Cd, 27-Cd and 28-Cd sequencing.

Conclusions and Results:

07 31 – all of the colonies are successful.
08 10 – all biobricks are sequenced succesfully.

About

pLac-cI repressor+ pLux/Ci-LuxI

Date Materials Experiment
07 19 pLac (R0010)
cI lambda repressor (P0151)		 pLac biobrick was cleaved with EcoRI and SpeI, L10 was constructed.
cI Lambda repressor biobrick was digested with EcoRI and XbaI + FastAP, L11 was constructed.
07 19 L10
L11		 L11 and L10 were ligated and transformed to DH5α.
07 20 Colony PCR was performed with VF2 and VR primers.
1st and 2nd colonies are inoculated. P18 is produced.
07 21 P14.2
P18		 P14.2 is digested with EcoRI and XbaI + Fast AP.
P18 is digested with EcoRI + SpeI.
07 21 Restriction products are ligated and transformed into DH5α competent cells.
07 22 Colony PCR was performed with VF2 and VR primers.
1st and 2nd colonies are inoculated. P48 is produced and sequenced.

Conclusions and Results:

07 25 - P48 is sequenced successfully.

About

Cd21L26 - Cascade with pLux/cI right promoter and strong RBS
Cd21L27 - Cascade with pLux/cI right promoter and Medium RBS
Cd21L28 - Cascade with pLux/cI right promoter and weak RBS

Date Materials Experiment
08 26 BL21-DE3
Cd21L26
Cd21L27
Cd21L28
PET-HCsy3		 Transformation of Cd21L26 and PET-HCsy3 plasmids into BL21-DE3 bacteria.
Transformation of Cd21L27 and PET-HCsy3 plasmids into BL21-DE3 bacteria.
Transformation of Cd21L28 and PET-HCsy3 plasmids into BL21-DE3 bacteria.
08 27 BL21-DE3
Cd21L26
Cd21L27
Cd21L28
PET-HCsy3		 Transformation of Cd21L26 plasmid into BL21-DE3 bacteria.
Transformation of Cd21L27 plasmid into BL21-DE3 bacteria.
Transformation of Cd21L28 plasmid into BL21-DE3 bacteria.
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria.
08 28 BL21-DE3 transformants
Cd21L26
Cd21L27
Cd21L28
PET-HCsy3 		Transformation of Cd21L26 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Transformation of Cd21L27 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Transformation of Cd21L28 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L26).
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L27).
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L28).
08 29 BL21-DE3 transformants Growth of transformants in liquid LB.
Preparing to repeat transformation.
08 30 BL21-DE3 transformants
Cd21L26
Cd21L27
Cd21L28
PET-HCsy3		 Transformation of Cd21L26 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Transformation of Cd21L27 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Transformation of Cd21L28 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L26).
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L27).
Transformation of and PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L28).
08 31 BL21-DE3 transformants Repeating transformation for better results.
Inoculating night culture of transformants.
09 01 BL21-DE3 transformants
Cd21L26
Cd21L27
Cd21L28
PET-HCsy3		 Repeating transformation of Cd21L26 and PET-HCsy3 plasmids into BL21-DE3 bacteria, Cd21L27 and PET-HCsy3 and Cd21L28 and PET-HCsy3 into BL21-DE3 bacteria.
Repeating transformation of Cd21L26, Cd21L27 and Cd21L28 plasmid into BL21-DE3 bacteria (transformed with PET-HCsy3).
Repeating transformation of PET-HCsy3 plasmid into BL21-DE3 bacteria (transformed with Cd21L26, Cd21L27 and Cd21L28).
Trying to improve transformation by putting less antibiotics on a Petri dish.
09 02 BL21-DE3 transformants Checking viability of the colonies by inoculating them in liquid LB for a few hours.
09 03 BL21-DE3 transformants Transformants are inoculated into 20 mL LB and grown for 3 hours at 37ºC.
Half of the transformants are put into 16ºC and grown overnight.
Other half is centrifuged, supernatant discarded and cells put into 4ºC for further experiments.
09 04 BL21-DE3 transformants
Depleting buffer
PMSF
Protein Dye		 Transformants from 16ºC are centrifuged, supernatant discarded.
Lysing all transformant cells with ultrasound.
Lysate transferred into fresh collection tubes (1.5 mL) and used for Western Blot samples preparation.
09 07 Polyacrylamid gel
Protein samples		 SDS-PAGE is made in order to decide the right volume of protein solution for Western Blot.
09 08 Polyacrylamid gel
PVDF membraine
Primary antibodies		 Doing Western Blot
Incubating with primary antibodies overnight
09 10 Secondary antibodies Finishing Western Blot
Incubating with secondary antibodies
Visualizing blot results

Conclusions and Results:

08 27 – Transformation unsuccessful.
08 28 – Transformation successful.
08 29 – Few colonies grown, but none multiplied in liquid LB with antibiotics.
08 31 – Few colonies have grown on a plate.
09 01 – Night culture did not grow. Transformation unsuccessful.
09 02 – Few colonies have grown, but all viable.
09 04 – Western Blot samples were made.
09 07 – Decided what volume of protein should be used for western blot.
09 10 – Visualized Western Blot results.

About

Cd21L26 - Cascade with pLux/cI right promoter and strong RBS
Cd21L27 - Cascade with pLux/cI right promoter and Medium RBS
Cd21L28 - Cascade with pLux/cI right promoter and weak RBS

Date Materials Experiment
09 04 SP1
SP3
pAcYc plasmid
DH5α
Karbenicilin		 Restriction of pCDF vector, SP1 and SP3 genes with EcoRI and XbaI.
Ligating pieces afterwards, transforming new pCDF plasmid into bacteria.
09 05 DH5α transformants
Bl21-DE3 transformants
pCola-Cas1-Cas3 plasmid
Chloramphenicol
Karbenicilin 		Colony PCR. Inoculating good colonies.
Inoculating Bl21-DE3 transformants (BL21-DE3 with Cd21L26 + HCsy3; Cd21L27 + HCsy3; Cd21L28 + HCsy3) overnight.
09 06 Bl21-DE3 transformants
pCola-Cas1-Cas3 plasmid
SP1
SP3
pCDF
DH5α bacteria
Chloramphenicol
Kanamycin
Streptomycin 		Inoculating night culture of Bl21-DE3 transformants for 3 hours.
Preparing competent cells. Transforming pCola plasmid into BL21-DE3 transformants.
Repeat cloning of SP1 and SP3 into pCDF vector and transforming them into DH5α.
09 07 DH5α transformants
Bl21-DE3 transformants
pCola-Cas1-Cas3 plasmid
Chloramphenicol
Karbenicilin
Kanamycin
Streptomycin 		Colony PCR. Inoculating good SP1 and SP3 colonies overnight. Repeating yesterdays transformation. Inoculating night colonies again, in case of transformation failure.
09 08 DH5α transformants
Bl21-DE3 transformants
pCola-Cas1-Cas3 plasmid
Chloramphenicol
Karbenicilin
Kanamycin 		Extracting SP1 and SP3 plasmids. Repeating transformation.
09 09 Bl21-DE3 bacteria
pCola-Cas1-Cas3 plasmid
Cd21L26 plasmid
Cd21L27 plasmid
Cd21L28 plasmid
Chloramphenicol
Kanamycin 		Transformation of pCola and Cd21L26 into BL21-DE3 bacteria.
Transformation of pCola and Cd21L27 into BL21-DE3 bacteria.
Transformation of pCola and Cd21L28 into BL21-DE3 bacteria.
09 10 BL21-DE3 transformants
Chloramphenicol
Kanamycin 		Inoculating transformants overnight.
09 11 BL21-DE3 transformants
pCDF-SP1 plasmid
pCDF-SP3 plasmid
Chloramphenicol
Kanamycin
Streptomycin		 Measuring night culture optical dense. Equalizing optical dense of all bacteria.
Preparing cells for transformation. Transforming pCDF-SP1, pCDF-SP3 and pCDF plasmids into BL21-DE3 transformants.
09 12 BL21-DE3 transformants
pCDF-SP1 plasmid
pCDF-SP3 plasmid
Chloramphenicol
Kanamycin
Streptomycin		 Repeating transformation. Before inoculating bacteria on a Petri dish, it is diluted 100 and 10000 times.
09 13 BL21-DE3 transformants
pCDF-SP1 plasmid
pCDF-SP3 plasmid
Chloramphenicol
Kanamycin
Streptomycin		 Repeating transformation.
Before inoculating bacteria on a petri dish, it is diluted 100 and 1000 times.
09 14 BL21-DE3 transformants
pCDF-SP1 plasmid
pCDF-SP3 plasmid
Chloramphenicol
Kanamycin
Streptomycin		 Repeating transformation.
Before bacteria inoculation on a petri dish, it is diluted 100 and 10000 times.
09 15 BL21-DE3 transformants
Chloramphenicol
Kanamycin		 Inoculating new BL21-DE3 transformant cells. Growing overnight.
09 16 BL21-DE3 transformants
pCDF-SP1 plasmid
pCDF-SP3 plasmid
Chloramphenicol
Kanamycin
Streptomycin		 Repeating transformation. Before bacteria inoculation on a petri dish, it is diluted 100 and 10000 times.
09 17 BL21-DE3 transformants Counting grown colonies.

Conclusions and Results:

09 05 – Transformation successful. Colony PCR showed, that SP1 and SP3 fragments were cloned successfully.
09 06 – Mistake found. SP1 and SP3 were cloned into a wrong vector.
09 07 – Transformation was unsuccessful.
09 08 – Transformation unsuccessful. Trying to reduce antibiotic quantity.
09 09 – Transformation unsuccessful. PET-HCsy3 plasmid may have inhibited transformation.
09 10 – Transformation successful.
09 11 – BL21-DE3 transformed with Cd21L27+pCola did not grow.
09 12 – Too much bacteria grew up.
09 13 – Transformation unsuccessful. In some dishes still too many bacteria grown.
09 14 – Transformation unsuccessful.
09 15 – Transformation unsuccessful. Probably not enough pCDF-SP1 and pCDF-SP3 vector.
09 17 - Transformation successful.