Difference between revisions of "Team:Exeter/Labbook"

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  The T5 promoter (BBa_K592008), Double terminator (BBa_B0015), RBS (BBa_B0034), non codon optimized Killer Red (BBa_K1184000),
 
  The T5 promoter (BBa_K592008), Double terminator (BBa_B0015), RBS (BBa_B0034), non codon optimized Killer Red (BBa_K1184000),
 
  Strong, Medium and Weak Constitutive Promoters (BBa_J23101,BBa_J23110,BBa_J23103) resuspended DNA was then transformed,  
 
  Strong, Medium and Weak Constitutive Promoters (BBa_J23101,BBa_J23110,BBa_J23103) resuspended DNA was then transformed,  
−
  1μl of DNA was inoculated into DH5a competent cells and then the transformation protocol (1) was followed.  
+
  1μl of DNA was inoculated into DH5α competent cells and then the transformation protocol (1) was followed.  
 
  We plated on the corresponding antibiotic and LB plates and left the plates overnight in the non-shaking 37&#8451; incubator.</p>
 
  We plated on the corresponding antibiotic and LB plates and left the plates overnight in the non-shaking 37&#8451; incubator.</p>
 
<h2>14/07/16</h2>
 
<h2>14/07/16</h2>
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  Extension was run for 105 secs (3 x 30sec per kbp as pKD4 is 3267 bp, add 15 sec for extra time)
 
  Extension was run for 105 secs (3 x 30sec per kbp as pKD4 is 3267 bp, add 15 sec for extra time)
 
   
 
   
−
Transformed Q5 PCR products into DH5a <i>E. coli</i>.</p>
+
Transformed Q5 PCR products into DH5α <i>E. coli</i>.</p>
 
<h2>21/07/16</h2>
 
<h2>21/07/16</h2>
 
<h6>Team: Dan, Eloise, Jack</h6>
 
<h6>Team: Dan, Eloise, Jack</h6>
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  as one part and the Killer Red WT protein coding region with the terminator as a second part. These two initial parts must  
 
  as one part and the Killer Red WT protein coding region with the terminator as a second part. These two initial parts must  
 
  then be ligated to form the entire promoter, RBS, coding region and terminator sequence. Today's work involved the initial
 
  then be ligated to form the entire promoter, RBS, coding region and terminator sequence. Today's work involved the initial
−
  digestion and ligation using a tetracycline plasmid backbone. The PCR product produced was then transformed into DH5a</p>
+
  digestion and ligation using a tetracycline plasmid backbone. The PCR product produced was then transformed into DH5α</p>
 
<h2>22/07/16</h2>
 
<h2>22/07/16</h2>
 
<h6>Team: Jack, Eloise, Hannah, Dan, Joel, Emily</h6>
 
<h6>Team: Jack, Eloise, Hannah, Dan, Joel, Emily</h6>
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Q5- site directed Mutagenesis
 
Q5- site directed Mutagenesis
 
The PCR and gel electrophoresis were successful yesterday.  
 
The PCR and gel electrophoresis were successful yesterday.  
−
However, the PCR product was not successfully transformed into DH5.
+
However, the PCR product was not successfully transformed into DH5α.
 
  It was later discovered that this is due to the pKD4 plasmid being toxic to this strain of <i>E.coli</i>.  
 
  It was later discovered that this is due to the pKD4 plasmid being toxic to this strain of <i>E.coli</i>.  
 
All of the PCR product remaining was loaded onto the gel so the reaction had to be repeated.
 
All of the PCR product remaining was loaded onto the gel so the reaction had to be repeated.
−
  The second gel was successful so the PCR product was transformed into <i>E.coli</i> DH5a and incubated at 37&#8451;
+
  The second gel was successful so the PCR product was transformed into <i>E.coli</i> DH5α and incubated at 37&#8451;
 
  overnight. However this transformation was also unsuccessful due to toxicity of pKD4 to this <i>E.coli</i> strain.</p>
 
  overnight. However this transformation was also unsuccessful due to toxicity of pKD4 to this <i>E.coli</i> strain.</p>
 
<h2>25/07/16</h2>
 
<h2>25/07/16</h2>
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Cas9 protein was resuspended using too much water from the 2016 distribution kit so the correct amount
 
Cas9 protein was resuspended using too much water from the 2016 distribution kit so the correct amount
−
  of resuspension was added to our left over 2015 distribution kit. This was then transformed into DH5a.</p>
+
  of resuspension was added to our left over 2015 distribution kit. This was then transformed into DH5α.</p>
  
 
<h2>26/07/16</h2>
 
<h2>26/07/16</h2>
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The QC multi kit was unsuccessful yesterday and so was repeated again today to remove multiple restriction  
 
The QC multi kit was unsuccessful yesterday and so was repeated again today to remove multiple restriction  
 
sites in the pKD4 plasmid.
 
sites in the pKD4 plasmid.
−
Overnights were made of the cloned KO/KR in DH5a.</p>
+
Overnights were made of the cloned KO/KR in DH5α.</p>
  
 
<h2>04/08/16</h2>
 
<h2>04/08/16</h2>
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<p id="pp">Glycerol stocks, mini preps and qubit was carried out on the Killer Orange and Killer red overnights in S171.
 
<p id="pp">Glycerol stocks, mini preps and qubit was carried out on the Killer Orange and Killer red overnights in S171.
−
Overnights for successfully transformed ccdB plasmid backbones and KO/KR in S171 and DH5a were made.</p>
+
Overnights for successfully transformed ccdB plasmid backbones and KO/KR in S171 and DH5α were made.</p>
  
 
<h2>05/08/16</h2>
 
<h2>05/08/16</h2>
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<p id="pp">Glycerol stocks, mini prep and qubit of ccdB plasmid backbones as well as KO and KR from MoClo in S171  
 
<p id="pp">Glycerol stocks, mini prep and qubit of ccdB plasmid backbones as well as KO and KR from MoClo in S171  
−
and DH5a strains were carried out before sending these for sequencing.
+
and DH5α strains were carried out before sending these for sequencing.
 
The QC multi was carried out again and the controls showed it to be successful by turning blue.</p>
 
The QC multi was carried out again and the controls showed it to be successful by turning blue.</p>
  
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  these were used for our mini experiments on reverse GFP.
 
  these were used for our mini experiments on reverse GFP.
 
  Transformations were also carried out for pKD4, KO and KR.
 
  Transformations were also carried out for pKD4, KO and KR.
−
Overnights of pSB1C3 and wild type DH5a were made to be used in the mini stat tomorrow.  
+
Overnights of pSB1C3 and wild type DH5α were made to be used in the mini stat tomorrow.  
 
Dan and supervisor Paul investigated the setup and control of the mini stat today ready to be used tomorrow
 
Dan and supervisor Paul investigated the setup and control of the mini stat today ready to be used tomorrow
 
  for initial testing.</p>
 
  for initial testing.</p>
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The mini stat was set up. Starting optical densities (OD) of the overnighted pSB1C3 was
 
The mini stat was set up. Starting optical densities (OD) of the overnighted pSB1C3 was
 
  taken before it was inoculated into the mini stat chambers.  
 
  taken before it was inoculated into the mini stat chambers.  
−
More overnights were made of the KO and KR as well as reverse GFP pJET products in DH5a.</p>
+
More overnights were made of the KO and KR as well as reverse GFP pJET products in DH5α.</p>
  
 
<h2>10/08/16</h2>
 
<h2>10/08/16</h2>
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  Qubits of these parts were carried out before MoClo was started to ascertain the exact volumes required  
 
  Qubits of these parts were carried out before MoClo was started to ascertain the exact volumes required  
 
  according to the DNA part concentration.
 
  according to the DNA part concentration.
−
Transformations of KR3 into BL21DE3, DNase and lysozyme into DH5a.  
+
Transformations of KR3 into BL21DE3, DNase and lysozyme into DH5α.  
 
The OD of the continuous cultures in the mini stat were taken. The burettes were emptied and the flow rate was lowered  
 
The OD of the continuous cultures in the mini stat were taken. The burettes were emptied and the flow rate was lowered  
 
to reduce the amount of effluence produced overnight.
 
to reduce the amount of effluence produced overnight.
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<h6>Team: Dan, Eloise, Jack, Pablo</h6>
 
<h6>Team: Dan, Eloise, Jack, Pablo</h6>
  
−
<p id="pp">DNase and lysozyme were cloned again then transformed into DH5a.
+
<p id="pp">DNase and lysozyme were cloned again then transformed into DH5α.
 
  Killer red overnights were glycerol stocked, mini prepped and quibitted.
 
  Killer red overnights were glycerol stocked, mini prepped and quibitted.
 
  Flasks were set up to test Killer red and killer orange again.</p>
 
  Flasks were set up to test Killer red and killer orange again.</p>
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these reagents to be stored for later work. We transformed Killer Orange, Killer Red and Lysozyme  
 
these reagents to be stored for later work. We transformed Killer Orange, Killer Red and Lysozyme  
 
into BL21 DE3 <i>E.coli</i> and started competent cell prep of this strain. Later in the day overnights were  
 
into BL21 DE3 <i>E.coli</i> and started competent cell prep of this strain. Later in the day overnights were  
−
made to be used for testing tomorrow for an SDS page gel, these included Killer Red BL21DE3 and wild type DH5a.
+
made to be used for testing tomorrow for an SDS page gel, these included Killer Red BL21DE3 and wild type DH5α.
 
  The DNase was again cloned using the MoClo method and placed in the PCR machine overnight.  
 
  The DNase was again cloned using the MoClo method and placed in the PCR machine overnight.  
−
  We prepared a streak plate of DH5a Killer Orange to be sent to Glasgow iGEM team for a collaboration.</p>
+
  We prepared a streak plate of DH5α Killer Orange to be sent to Glasgow iGEM team for a collaboration.</p>
  
 
<h2>23/08/16</h2>
 
<h2>23/08/16</h2>
 
<h6>Team: Dan, Eloise, Jack</h6>
 
<h6>Team: Dan, Eloise, Jack</h6>
  
−
<p id="pp">MoClo DNase was removed from the PCR machine and transformed into DH5a.  
+
<p id="pp">MoClo DNase was removed from the PCR machine and transformed into DH5α.  
−
In addition to this KR3 was also transformed into DH5a. Overnights were made of all transformations from yesterday as all
+
In addition to this KR3 was also transformed into DH5α. Overnights were made of all transformations from yesterday as all
 
  were successful. The lysozyme horizontal gene transfer experiment was performed (see lysozyme HGT protocol)
 
  were successful. The lysozyme horizontal gene transfer experiment was performed (see lysozyme HGT protocol)
 
  and left overnight in the PCR machine at 55?.
 
  and left overnight in the PCR machine at 55?.
−
5ml samples were taken from the culture flasks of KR induced, KR not induced and WT DH5a and put into the cold room. </p>
+
5ml samples were taken from the culture flasks of KR induced, KR not induced and WT DH5α and put into the cold room. </p>
  
 
<h2>24/08/16</h2>
 
<h2>24/08/16</h2>
Line 1,001: Line 1,001:
 
  and 0.7ml of LB. The left over lysate was plated out as a control to see if the cells survived the reaction.  
 
  and 0.7ml of LB. The left over lysate was plated out as a control to see if the cells survived the reaction.  
 
  The EnzChek standard curve was performed. Competent BL21 DE3 cells were made and put into the -80&#8451; freezer.
 
  The EnzChek standard curve was performed. Competent BL21 DE3 cells were made and put into the -80&#8451; freezer.
−
  An SDS page gel was performed on the 4 hr and 20 hr induced KR, and WT DH5a,
+
  An SDS page gel was performed on the 4 hr and 20 hr induced KR, and WT DH5α,
 
  it didn't show any real difference. Overnights of the successful DNase plates were made.  </p>
 
  it didn't show any real difference. Overnights of the successful DNase plates were made.  </p>
  
Line 1,011: Line 1,011:
 
  This is an initial indication that HGT of DNA from a cell that has undergone lysis using production of lysozyme  
 
  This is an initial indication that HGT of DNA from a cell that has undergone lysis using production of lysozyme  
 
  can in principle happen. More repeats of the experiment will be undertaken.
 
  can in principle happen. More repeats of the experiment will be undertaken.
−
  The overnight cultures of KO BL21 DE3, KR BL21 DE3 and pSB1C3 RFP DH5a were used to make 4.5ml
+
  The overnight cultures of KO BL21 DE3, KR BL21 DE3 and pSB1C3 RFP DH5α were used to make 4.5ml
 
  samples at a dilution factor of 10-3,10-4 and 10-5. One set of fifteen samples  
 
  samples at a dilution factor of 10-3,10-4 and 10-5. One set of fifteen samples  
 
  (see Light Box protocol for samples) was exposed to light in the light box. A duplicate set of samples was kept  
 
  (see Light Box protocol for samples) was exposed to light in the light box. A duplicate set of samples was kept  
Line 1,027: Line 1,027:
 
CFU's were counted on the spread plates from the light exposure experiment.  
 
CFU's were counted on the spread plates from the light exposure experiment.  
 
Concentrations of the DNAse miniprep was measured so this can now be sent for sequencing.  
 
Concentrations of the DNAse miniprep was measured so this can now be sent for sequencing.  
−
Glycerol stocks were made of the KR3 in DH5a and lysozyme in BL21 DE3
+
Glycerol stocks were made of the KR3 in DH5α and lysozyme in BL21 DE3
 
</p>
 
</p>
  

Revision as of 19:55, 18 October 2016