Difference between revisions of "Team:British Columbia/Notebook"

Line 488: Line 488:
 
          
 
          
 
<br>
 
<br>
 +
 
    
 
    
 
F3H
 
F3H
Line 517: Line 518:
 
<br>
 
<br>
 
<b> Parts we've synthesized ourselves: </b>
 
<b> Parts we've synthesized ourselves: </b>
 +
</p>
  
 +
<p>
 
<br>
 
<br>
  
Line 524: Line 527:
 
~1011 BP
 
~1011 BP
 
pSB1C3 (Cm)
 
pSB1C3 (Cm)
−
 
 
</p>
 
</p>
  
−
<br>
+
<h3 style="margin-left:70px;">July</h3>
−
<p>First, combining F3H and B0034:</p>
+
  <ul>
−
<ul>
+
    <li>26:
−
<li> Transform from distribution kit wells via heat shock method</li>
+
      <ul>
−
<li> Spread on CMB-LB agar plate at 37c° overnight
+
        <li>Transformed F3H and promoter+rbs into DH5a</li>
−
<li> Pick and inoculate with LB+Cm 37c° for 16 hours </li>
+
        <li>Transformed GFP + RBS into DH5a</li>
−
<li> Miniprep to obtain plasmids (elution buffer, 50μm) </li>
+
      </ul>
−
<li> Digest F3H with XbaI + SpeI and B0034 with EcoRI and XbaI </li>
+
    </li>
−
<li> Gel electrophoresis and gel purify </li>
+
−
<li> Ligate F3H and B0034 together with T4 ligase at room temperature for 1 hour </li>
+
−
<li> Transform via heat shock method </li>
+
−
<li> Spread on CMB-LB agar plate at 37c° overnight
+
−
<li> Pick and inoculate with LB+Cm 37c° for 16 hours </li>
+
−
<li> Miniprep to obtain plasmids (elution buffer, 50μm) </li>
+
−
<li> Plasmid obtained</li>
+
−
</ul>
+
  
−
<br>
+
    <li>27:
−
<p>Second, the FLS1 and: </p>
+
      <ul>
−
<ul>
+
        <li>Inoculated RFP + RBS in 25mg/uL of CM-25</li>
−
<li>CHS digest with EcoRI and SpeI </li>
+
        <li>Inoculated GFP + RBS in 25mg/uL of CM-25</li>
−
<li> CHI digest with EcoRI and Xbal </li>
+
      </ul>
−
<li> Ligation of both digestion with T4 ligase </li>
+
    </li>
−
<li> E. coli transformation via heat shock method </li>
+
−
<li> Spread on CMB-LB agar plate at 37c° overnight </li>
+
−
<li> PCR and gel electrophoresis to analyze if successful </li>
+
−
<li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°</li>
+
−
<li> Plasmid obtained</li>
+
−
</ul>
+
  
−
<br>
+
    <li>28:
−
<p>Third, combine the two plasmids previously prepared:</p>
+
      <ul>
−
<ul>
+
        <li>Miniprepped RFP + RBS</li>
−
<li> 4CL – TAL digest by EcoRI and SpeI</li>
+
        <li>Miniprepped GFP + RBS</li>
−
<li>CHS-CHI with EcoRI and Xbal </li>
+
        <li>Digested RFP + RBS  with ECOR1 and XBA1 to obtain 8.96uL of DNA at 111.16ng/uL</li>
−
<li> Dephosphorylate CHS-CHI </li>
+
        <li>Digested GFP + RBS with ECOR1 and XBA1 to obtain 12.8uL of DNA at 78.1ng/uL</li>
−
<li> Ligate the two together with T4 ligation </li>
+
        <li>Eventually discarded both digests as we did not use them</li>
−
<li> Add GFP via digestion and then ligation* </li>
+
      </ul>
−
<li> E. coli transformation via heat shock method </li>
+
    </li>
−
<li> PCR and gel electrophoresis to analyze if successful </li>
+
  </ul>
−
<li> Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°</li>
+
  <hr>
−
</ul>
+
  <h3 style="margin-left:70px;">August</h3>
 +
  <ul>
 +
    <li>7:
 +
      <ul>
 +
        <li>Transformed Bba_E0020 into DH5a</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>8:
 +
      <ul>
 +
        <li>Inoculated CFP strains with  25mg/uL of CM-25</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>9:
 +
      <ul>
 +
        <li>Digested CFP with ECOR1 + XBA1 to obtain 8.77uL of DNA at 114.0ng/uL</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>15:
 +
      <ul>
 +
        <li>Transformed RFP into DH5a</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>16:
 +
      <ul>
 +
        <li>Inoculated RFP in 25mg/uL of CM-25</li>
 +
        <li>Miniprepped and digested RFP with ECOR1 + XBA1 to obtain 9.14uL of DNA at 109.4ng/uL</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>18:
 +
      <ul>
 +
        <li>Transformed Bba_E0040 (GFP) into DH5a</li>
 +
        <li>Transformed Bba_l15016 (CFP + RBS) into DH5a</li>
 +
        <li>Synthesized parts from IDT were resuspended in 1X TBE</li>
 +
        <ul>
 +
          <li>FdeR</li>
 +
          <li>Pt181-repressor</li>
 +
          <li>Pt181-GP2</li>
 +
          <li>GP2</li>
 +
          <li>Antisense</li>
 +
        </ul>
 +
        <li>RFP, RFP+RBS, CFP, GFP+RBS have all been digested</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>19:
 +
      <ul>
 +
        <li>Transformed Bba_E0040 (GFP) into DH5a</li>
 +
        <li>Transfroemd CFP + RBS  into DH5a</li>
 +
        <li>Both transformations were unsuccessful</li>
 +
      </ul>
 +
    </li>
 +
 
 +
 
 +
    <li>20
 +
      <ul>
 +
        <li>OD of DH5a competent cells was 0.69</li>
 +
        <li>Pelleted competent cells using 0.1M CaCl2</li>
 +
        <li>Transformed Bba_E0040 (GFP) into DH5a</li>
 +
        <li>Transformed Bba_I5016 (CFP + RBS) into DH5a</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>21:
 +
      <ul>
 +
        <li>OD of DH5a competent cells was 0.53</li>
 +
        <li>Pelleted competent cells using 0.1M CaCl2</li>
 +
        <li>Positive and Negative controls did not function properly</li>
 +
        <li>Realized glycerol was not added</li>
 +
        <li>Re-setup experiment</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>23:
 +
      <ul>
 +
        <li>Reaction cleanup using ABM kit, involving:</li>
 +
        <ul>
 +
          <li>GP2</li>
 +
          <li>E+S</li>
 +
          <li>Pt181-GP2</li>
 +
          <li>FdeR</li>
 +
        </ul>
 +
        <li>Transformed Bba_E0040 (GFP) into DH5a</li>
 +
        <li>Transformed Bba_I15016 (CFP + RBS) into DH5a</li>
 +
        <li>Both transformations were unsuccessful</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>24
 +
      <ul>
 +
        <li>Ran plasmid PSB1C3 digested with X+P, with 1X TAE buffer</li>
 +
        <li>Ran plasmid PSB1C3 digested with E+S, with 1X TAE buffer</li>
 +
        <li>Ran plasmid PSB1C3 digested with E+P, with 1X TAE buffer</li>
 +
        <li>Purified with ABM kit:</li>
 +
        <ul>
 +
          <li>X+P</li>
 +
          <li>E+P</li>
 +
          <li>E+S</li>
 +
          <li>CFP</li>
 +
          <li>GFP+RBS</li>
 +
          <li>RFP+RBS</li>
 +
          <li>RFP</li>
 +
        </ul>
 +
        <li>Inoculated CFP and GFP in 25mg/uL of CM-25</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>25:
 +
      <ul>
 +
        <li>Miniprepped CFP+RBS and GFP (concentration 149ng/uL)</li>
 +
      </ul>
 +
      <li>27:
 +
        <ul>
 +
          <li>Ligated FdeR and Repressor DNA into RFP vector</li>
 +
          <li>Ligated FdER and Repressor DNA into E+S vector</li>
 +
          <li>Ligated pt181-GP2 and GP2 DNA into E+P</li>
 +
          <li>Ligated antisense DNA into X+P</li>
 +
        </ul>
 +
      </li>
 +
 
 +
      <li>29:
 +
        <ul>
 +
          <li>Transformed in DH5a:
 +
            RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
 +
          </li>
 +
        </ul>
 +
      </li>
 +
 
 +
      <li>30:
 +
        <ul>
 +
          <li>Inoculated in 25mg/uL of CM25:
 +
            RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
 +
          </li>
 +
        </ul>
 +
      </li>
 +
 
 +
      <li>31:
 +
        <ul>
 +
          <li>Miniprepped:
 +
            RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
 +
          </li>
 +
        </ul>
 +
      </li>
 +
 
 +
    </ul>
 +
 
 +
    <hr>
 +
 
 +
    <h3 style="margin-left:70px;">Spetember</h3>
 +
    <li>4:
 +
      <ul>
 +
        <li>Digested:
 +
          RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
 +
        </li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>5:
 +
      <ul>
 +
        <li>Ran Gel Electrophoresis In 1X TBE buffer on:
 +
          RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
 +
        </li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>7:
 +
      <ul>
 +
        <li>Double checked digestions with BamH1 + ECOR1  + Pst1 of strains:
 +
          RFP - FdeR, E+S-FdeR
 +
        </li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>13:
 +
      <ul>
 +
        <li>Transformed RFP - FdeR2 into DHd5a</li>
 +
        <li>Digested GFP Vector using: E+P, E+X, E+S</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>20:
 +
      <ul>
 +
        <li>Ran Gel Electrophoresis on: GFP Vector + (E+P, E+X, E+S)</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>22:
 +
      <ul>
 +
        <li>Purified: GFP - E+P, GFP - E+S</li>
 +
        <li>Digested Repressor (from IDT) using E+S</li>
 +
        <li>Ligated repressor with E+S vector, pt181 GP2 and GP2 with E+P digested vector. Antisense ligated with X+P vector</li>
 +
      </ul>
 +
    </li>
 +
 
 +
    <li>23:
 +
      <ul>
 +
        <li>Inoculated: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig</li>
 +
      </ul>
 +
    </li>
  
 +
    <li>24:
 +
      <ul>
 +
        <li>Miniprepped: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig</li>
 +
      </ul>
 +
    </li>
  
−
<p>Protocol for cloning promoter-RBS-4CL in pSB1C3<p>
+
    <li>24:
−
A.      Restriction enzyme digest
+
      <ul>
−
1.      Prepare the master mix in a microfuge tube by combining the following:
+
        <li>Digested with E+P: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig</li>
−
Component
+
       </ul>
−
Volume to add (ul)
+
    </li>
−
10X NEB Buffer 2.1
+
−
6
+
−
NEB PstI restriction enzyme
+
−
4
+
−
Autoclaved distilled water
+
−
26
+
−
TOTAL
+
−
36
+
−
+
−
2.      Zip-spin the master mix to collect all liquid at the bottom of the tube
+
−
3.      With a pipette set to 25 ul, pipette each reaction up and down several times to mix
+
−
4.      Vortex the master mix thoroughly
+
−
5.      Zip-spin the master mix again
+
−
6.       Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix)
+
−
7.      Label the three tubes containing the aliquots as follows:
+
−
a.      P-RBS
+
−
b.      4CL
+
−
c.      PDC (positive digest control)
+
  
 +
    <li>25:
 +
      <ul>
 +
        <li>Performed Gel Electrophoresis of DNA strains: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig</li>
 +
      </ul>
 +
    </li>
 +
    <hr>
 
         </div>
 
         </div>
  

Revision as of 01:37, 18 October 2018

Naringenin Notebook

We kindly received the following from UBC iGEM distribution kit of 2018:
Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
4CL BBa_K801093 ~1700 BP Spring 2018 Distribution Plate 2, Well 17D pSB1C3 (Cm)
RBS+TAL BBa_K1033000 ~1600 Spring 2018 Distribution Plate 4, Well 6C pSB1C3 (Cm)


First, TAL and 4CL with RBS (ribosomal binding site) to combine them:

  • 4CL digest with EcoRI + SpeI
  • TAL digest with EcoRI + Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate and incubate at 37 C° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
  • Plasmid obtained

Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):

  • CHS digest with EcoRI and SpeI
  • CHI digest with EcoRI and Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate at 37c° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
  • Plasmid obtained

Third, combine the two plasmids previously prepared:

  • 4CL – TAL digest by EcoRI and SpeI
  • CHS-CHI with EcoRI and Xbal
  • Dephosphorylate CHS-CHI
  • Ligate the two together with T4 ligation
  • Add GFP via digestion and then ligation*
  • E. coli transformation via heat shock method
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°

Protocol for cloning promoter-RBS-4CL in pSB1C3

A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)

Biosensor Notebook

July

  • 26:
    • Transformed RFP + RBS into DH5a
    • Transformed GFP + RBS into DH5a
  • 27:
    • Inoculated RFP + RBS in 25mg/uL of CM-25
    • Inoculated GFP + RBS in 25mg/uL of CM-25
  • 28:
    • Miniprepped RFP + RBS
    • Miniprepped GFP + RBS
    • Digested RFP + RBS with ECOR1 and XBA1 to obtain 8.96uL of DNA at 111.16ng/uL
    • Digested GFP + RBS with ECOR1 and XBA1 to obtain 12.8uL of DNA at 78.1ng/uL
    • Eventually discarded both digests as we did not use them

August

  • 7:
    • Transformed Bba_E0020 into DH5a
  • 8:
    • Inoculated CFP strains with 25mg/uL of CM-25
  • 9:
    • Digested CFP with ECOR1 + XBA1 to obtain 8.77uL of DNA at 114.0ng/uL
  • 15:
    • Transformed RFP into DH5a
  • 16:
    • Inoculated RFP in 25mg/uL of CM-25
    • Miniprepped and digested RFP with ECOR1 + XBA1 to obtain 9.14uL of DNA at 109.4ng/uL
  • 18:
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transformed Bba_l15016 (CFP + RBS) into DH5a
    • Synthesized parts from IDT were resuspended in 1X TBE
      • FdeR
      • Pt181-repressor
      • Pt181-GP2
      • GP2
      • Antisense
    • RFP, RFP+RBS, CFP, GFP+RBS have all been digested
  • 19:
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transfroemd CFP + RBS into DH5a
    • Both transformations were unsuccessful
  • 20
    • OD of DH5a competent cells was 0.69
    • Pelleted competent cells using 0.1M CaCl2
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transformed Bba_I5016 (CFP + RBS) into DH5a
  • 21:
    • OD of DH5a competent cells was 0.53
    • Pelleted competent cells using 0.1M CaCl2
    • Positive and Negative controls did not function properly
    • Realized glycerol was not added
    • Re-setup experiment
  • 23:
    • Reaction cleanup using ABM kit, involving:
      • GP2
      • E+S
      • Pt181-GP2
      • FdeR
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transformed Bba_I15016 (CFP + RBS) into DH5a
    • Both transformations were unsuccessful
  • 24
    • Ran plasmid PSB1C3 digested with X+P, with 1X TAE buffer
    • Ran plasmid PSB1C3 digested with E+S, with 1X TAE buffer
    • Ran plasmid PSB1C3 digested with E+P, with 1X TAE buffer
    • Purified with ABM kit:
      • X+P
      • E+P
      • E+S
      • CFP
      • GFP+RBS
      • RFP+RBS
      • RFP
    • Inoculated CFP and GFP in 25mg/uL of CM-25
  • 25:
    • Miniprepped CFP+RBS and GFP (concentration 149ng/uL)
  • 27:
    • Ligated FdeR and Repressor DNA into RFP vector
    • Ligated FdER and Repressor DNA into E+S vector
    • Ligated pt181-GP2 and GP2 DNA into E+P
    • Ligated antisense DNA into X+P
  • 29:
    • Transformed in DH5a: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 30:
    • Inoculated in 25mg/uL of CM25: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 31:
    • Miniprepped: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense

Spetember

  • 4:
    • Digested: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 5:
    • Ran Gel Electrophoresis In 1X TBE buffer on: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 7:
    • Double checked digestions with BamH1 + ECOR1 + Pst1 of strains: RFP - FdeR, E+S-FdeR
  • 13:
    • Transformed RFP - FdeR2 into DHd5a
    • Digested GFP Vector using: E+P, E+X, E+S
  • 20:
    • Ran Gel Electrophoresis on: GFP Vector + (E+P, E+X, E+S)
  • 22:
    • Purified: GFP - E+P, GFP - E+S
    • Digested Repressor (from IDT) using E+S
    • Ligated repressor with E+S vector, pt181 GP2 and GP2 with E+P digested vector. Antisense ligated with X+P vector
  • 23:
    • Inoculated: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 24:
    • Miniprepped: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 24:
    • Digested with E+P: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 25:
    • Performed Gel Electrophoresis of DNA strains: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig

    • Kaempferol Notebook

    We kindly received the following from UBC iGEM distribution kit of 2018:
    Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
    F3H BBa_K1497009 ~1107 BP Spring 2018 Distribution Plate 2, Well 12I pSB1C3 (Cm)
    RBS BBa_B0034 ~12 BP Spring 2018 Distribution Plate 4, Well 1N pSB1C3 (Cm)
    Double Terminator BBa_B0015 ~129 BP Spring 2018 Distribution Plate 3, Well 3F pSB1C3 (Cm)
    Parts we've synthesized ourselves:


    FLS1 BBa_K2700000 ~1011 BP pSB1C3 (Cm)

    July

    • 26:
      • Transformed F3H and promoter+rbs into DH5a
      • Transformed GFP + RBS into DH5a
    • 27:
      • Inoculated RFP + RBS in 25mg/uL of CM-25
      • Inoculated GFP + RBS in 25mg/uL of CM-25
    • 28:
      • Miniprepped RFP + RBS
      • Miniprepped GFP + RBS
      • Digested RFP + RBS with ECOR1 and XBA1 to obtain 8.96uL of DNA at 111.16ng/uL
      • Digested GFP + RBS with ECOR1 and XBA1 to obtain 12.8uL of DNA at 78.1ng/uL
      • Eventually discarded both digests as we did not use them

    August

    • 7:
      • Transformed Bba_E0020 into DH5a
    • 8:
      • Inoculated CFP strains with 25mg/uL of CM-25
    • 9:
      • Digested CFP with ECOR1 + XBA1 to obtain 8.77uL of DNA at 114.0ng/uL
    • 15:
      • Transformed RFP into DH5a
    • 16:
      • Inoculated RFP in 25mg/uL of CM-25
      • Miniprepped and digested RFP with ECOR1 + XBA1 to obtain 9.14uL of DNA at 109.4ng/uL
    • 18:
      • Transformed Bba_E0040 (GFP) into DH5a
      • Transformed Bba_l15016 (CFP + RBS) into DH5a
      • Synthesized parts from IDT were resuspended in 1X TBE
        • FdeR
        • Pt181-repressor
        • Pt181-GP2
        • GP2
        • Antisense
      • RFP, RFP+RBS, CFP, GFP+RBS have all been digested
    • 19:
      • Transformed Bba_E0040 (GFP) into DH5a
      • Transfroemd CFP + RBS into DH5a
      • Both transformations were unsuccessful
    • 20
      • OD of DH5a competent cells was 0.69
      • Pelleted competent cells using 0.1M CaCl2
      • Transformed Bba_E0040 (GFP) into DH5a
      • Transformed Bba_I5016 (CFP + RBS) into DH5a
    • 21:
      • OD of DH5a competent cells was 0.53
      • Pelleted competent cells using 0.1M CaCl2
      • Positive and Negative controls did not function properly
      • Realized glycerol was not added
      • Re-setup experiment
    • 23:
      • Reaction cleanup using ABM kit, involving:
        • GP2
        • E+S
        • Pt181-GP2
        • FdeR
      • Transformed Bba_E0040 (GFP) into DH5a
      • Transformed Bba_I15016 (CFP + RBS) into DH5a
      • Both transformations were unsuccessful
    • 24
      • Ran plasmid PSB1C3 digested with X+P, with 1X TAE buffer
      • Ran plasmid PSB1C3 digested with E+S, with 1X TAE buffer
      • Ran plasmid PSB1C3 digested with E+P, with 1X TAE buffer
      • Purified with ABM kit:
        • X+P
        • E+P
        • E+S
        • CFP
        • GFP+RBS
        • RFP+RBS
        • RFP
      • Inoculated CFP and GFP in 25mg/uL of CM-25
    • 25:
      • Miniprepped CFP+RBS and GFP (concentration 149ng/uL)
    • 27:
      • Ligated FdeR and Repressor DNA into RFP vector
      • Ligated FdER and Repressor DNA into E+S vector
      • Ligated pt181-GP2 and GP2 DNA into E+P
      • Ligated antisense DNA into X+P
    • 29:
      • Transformed in DH5a: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
    • 30:
      • Inoculated in 25mg/uL of CM25: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
    • 31:
      • Miniprepped: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense

    Spetember

  • 4:
    • Digested: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 5:
    • Ran Gel Electrophoresis In 1X TBE buffer on: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 7:
    • Double checked digestions with BamH1 + ECOR1 + Pst1 of strains: RFP - FdeR, E+S-FdeR
  • 13:
    • Transformed RFP - FdeR2 into DHd5a
    • Digested GFP Vector using: E+P, E+X, E+S
  • 20:
    • Ran Gel Electrophoresis on: GFP Vector + (E+P, E+X, E+S)
  • 22:
    • Purified: GFP - E+P, GFP - E+S
    • Digested Repressor (from IDT) using E+S
    • Ligated repressor with E+S vector, pt181 GP2 and GP2 with E+P digested vector. Antisense ligated with X+P vector
  • 23:
    • Inoculated: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 24:
    • Miniprepped: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 24:
    • Digested with E+P: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 25:
    • Performed Gel Electrophoresis of DNA strains: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig

    • Plasmid Maintenance Notebook