Team:Imperial College London/Lab Diaries/XylE team

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{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
-
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|XylE Team
+
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"| XylE Team  
|-
|-
|
|
-
===Week 6===
+
'''Objectives:'''
 +
*construction of XylE fusion protein
 +
*Testing expression of XylE in E.coli and characterization under the control of a constitutive promoter
 +
*Construction of the -ComE promoter/XylE fussion protein- expression system
 +
*Construction of the -LacI promoter/XylE fusion construct- expression system
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
 
-
|-
+
'''Here's a picture of the final construct:'''
-
  ! Week 6 !! Monday, 9th-Aug-2010 !! Tuesday, 10th-Aug-2010!! Wednesday, 11th-Aug-2010 !! Thursday, 12th-Aug-2010 !! Friday, 13th-Aug-2010
+
 
-
|-
+
[[Image:IC_Module3.JPG|center]]
-
  | '''MORNING''' || || ||  || ||
+
|}
-
* Gel electrophoresis of pSB1C3 PCR product between EcoRI site and PstI
+
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
-
* PCR Purification of pSB1C3
+
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Typical XylE assay
|-
|-
-
  | '''AFTERNOON''' ||  || || ||
+
|align="center"|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/lyBMpN6zuHw?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/lyBMpN6zuHw?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html>
-
 
+
-
 
+
-
||
+
-
 
+
-
* Restriction digest of pSB1C3 and pSB1AK3 with EcoRI and PstI.  
+
-
 
+
|}
|}
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"| Week 6
 +
|-
 +
|
 +
<html>
 +
<table width="800px" border="0">
 +
  <tr>
 +
    <td style="background-color:#FFFF66;text-align:center;color:#555555;">
 +
      <b>Week 6</b>
 +
    </td> 
 +
<td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Monday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Tuesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Wednesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Thursday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Friday</b>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="background-color:#FFCC66;width:50px;text-align:center;color:#555555;"><b>Morning</b>
 +
    </td>
 +
<td style="background-color:#eeeeee;height:50px;width:100px;color:#555555;text-align:top;">
 +
    </td>
 +
<td style="background-color:#eeeeee;height:50px;width:100px;text-align:top;color:#555555;">
 +
    </td>
 +
<td style="background-color:#eeeeee;height:50px;width:100px;text-align:top;color:#555555;">
 +
    </td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:top;color:#555555;">   
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>mini-prep kit of XylE-transformed ''E.coli'' (already overnight grown)</li>
 +
</ul>
 +
</td>
 +
</tr>
 +
  <tr>
 +
<td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Afternoon</b>
 +
    </td>
 +
    <td style="background-color:#eeeeee;height:100px;width:100px;text-align:top;color:#555555;">
 +
    </td>
 +
<td style="background-color:#eeeeee;height:100px;width:100px;text-align:top;color:#555555;">
 +
    </td>
 +
<td style="background-color:#eeeeee;height:100px;width:100px;text-align:top;color:#555555;">
 +
    </td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Starting of “Testing expression of XylE in ''E.coli''” objective</li>
 +
<li>1)Annealing EcoRI and speI oligos to J23101 promoter which will be annealed later in front of the RBS-XylE registry gene (overnight) </li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>gel analysis of mini-prep derived XylE plasmid.(requires first digestion of the vector with restriction enzymes)</li>
 +
</ul> 
 +
</td>
 +
  </tr>
 +
</table>
 +
</html>
-
====Friday, 13th-Aug-2010====
 
-
'''Plan:'''
+
'''Thursday, 12-Aug-2010'''
-
*Perform gel electrophoresis to confirm that the pSB1C3 PCR between the EcoRI and the PstI site worked.
+
* Annealing DNA strands of J23101 promoter in a water bath
-
*If PCR worked pSB1C3 will be PCR purified
+
we constructed the standard ''E.coli'' promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assembly to construct a promoter-RBS-XylE design in a psB1C3 vector. ''E.coli'' will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted BioBricks.
-
*Restriction digest of purified pSB1C3 and pSB1AK3 with PstI and EcoRI
+
-
*Gel electrophoresis and gel purification of excised double terminator BOO14
+
-
*If possible, set up ligation over the weekend
+
-
'''Report:'''
+
* Prepared two overnight cultures of XylE transformed ''E.coli'' (one 50microliters and one of 450μl)
-
*Analzyzing the PCR product with gel electrophoresis, we observed -as expected- a 2kb fragment on the gel. This confimrs that the PCR of pSB1C3 was successful as the vector is approximately 2kb long.
+
these cultures are going to be used tomorrow for mini prepping. Mini prep will allow us to isolate ''E.coli'' 's plasmid DNA(which contains the XylE gene).
-
*Having confirmed that the PCR was successful we purified the PCR product from the solution using the ''E.Z.N.A.Æ Cycle Pure Kit (Omega bio-tek)'' but used 25µl of ddH2O instead of elusion buffer in the last step.
+
-
*We then did a restriction digestion for:
+
-
# The pSB1C3 PCR product using the enzymes PstI and EcoRI in buffer 4
+
-
# pSB1A3 with B0014 using the enzymes PstI and EcoRI also in buffer 4
+
-
*Our last step was to use gel electrophoresis to separate the product of the restriction digest to confirm successful digestion and purification. Unfortunately we lost the excised BOO14 during electrophoresis so digestion of pSB1AK3 has to be repeated.
+
-
====Saturday, 14th-Aug-2010====
+
'''Friday, 13-Aug-2010'''
-
 
+
* Mini-prep of XylE transformed ''E.coli''
-
'''Plan:'''
+
Mini prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasmid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini prep will later be used for the midi-prep (that gives out higher yields of DNA needed for cloning).
-
*Repeat digestion of pSB1AK3 with PstI and EcoRI
+
* Gel analysis of plasmid DNA retrieved from mini prep of XylE transformed ''E.coli'', cut with restriction enzymes. From light to the left, 50μg digested DNA : 50 undigested DNA : 450 digested DNA : 450 undigested DNA. In lanes 1 and 3 the smaller band has a size of about 1kB which corresponds to RBS-XylE gene. The bigger bands are the cut vectors. In lanes 2 and 4 is the uncut BioBrick from the registry. It appears smaller on the gel than it actually is as circular DNA travels faster through the pores of agarose gel rather than linearised DNA.
-
*Gel electrophoresis of the digestion products to separate BOO14 and the vector to isolate BOO14
+
|}
-
'''Report:'''
+
-
*Digestion of pSB1AK3 with PstI and EcoRI was performed to excise the terminator BOO14.
+
-
*Gel electrophoresis was successfully used to separate the digestion products pSB1Ak3 and BOO14, although yield of the later appears to be relatively low probably due to the short length of the terminator. The band of BOO14 was cut out of the gel.
+
-
 
+
-
 
+
-
 
+
-
===Week 7===
+
-
 
+
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
+
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"| Week 7
|-
|-
-
  ! Week 6 !! Monday, 16th-Aug-2010 !! Tuesday, 17th-Aug-2010!! Wednesday, 18th-Aug-2010 !! Thursday, 19th-Aug-2010 !! Friday, 20th-Aug-2010
+
|
-
|-
+
[[Image:IC_10-08-13_Digest_image.jpg|thumb|center|400px]]
-
  | '''MORNING''' ||  
+
-
* Gel purification of BOO14
+
<html>
-
* Determine concentrations of BOO14 and pSB1C3
+
<table width="850px" border="0">
-
* Restriction digest of pSB1C3 with EcoRI and PstI
+
  <tr>
 +
    <td style="background-color:#FFFF66;text-align:center;color:#555555;">
 +
      <b>Week 7</b>
 +
    </td>
 +
<td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Monday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Tuesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Wednesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Thursday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Friday</b>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Morning</b>
 +
    </td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>midi prep XylE ''E.coli'' (2hrs)</li>
 +
</ul>
 +
</td>
-
* ''Work on Wiki''
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>gel extraction kit on XylE gene trapped in agarose </li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>3A assemply: make replica plates (overnight)</li>
 +
<li>Catechol assay of ''E.coli'' </li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Mini prep XylE ''E.coli''</li>
 +
</ul>
 +
</td>
-
* Check plates with transformants for colonies
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* Perform colony PCR to confirm if ligation and transformation were successful
+
<ul>
-
* Prepare a replica plate
+
<li>Midi prep XylE ''E.coli''</li>
-
* Set up of pSB1C3-BOO14 cultures for mini preps
+
</ul>
 +
</td>
 +
</tr>
 +
  <tr>
 +
<td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Afternoon</b>
 +
    </td>
 +
 
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>restriction digestion of XylE for 3A assemply</li>
 +
<li>gel purification of XylE from restriction digestion</li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>3A assembly of vector, XylE and J23101 promoter </li>
 +
<li>above construct transformed in ''E.coli''</li>
 +
</ul>
 +
</td>
-
* Set up PCR reaction with potentially contaminated agents
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* Gel electrophoresis of PCR products to determine which component is contaminated with DNA
+
<ul>
-
* Mini prep to isolate pSB1C3-BOO14
+
<li>primers arrived!</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>PCR extension of XylE and GFP, round 1</li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
</td>
 +
</tr>
 +
</table>
 +
</html>
-
* Repeat gel electrophoresis of pSB1C3-BOO14 restriction digest
 
-
* Perform PCR to amlify CWB out of the B. ''subtilis'' genome
 
-
|-
+
'''Monday, 16-Aug-2010'''
-
  | '''AFTERNOON''' ||
+
* Midi prepped the XylE-transformed ''E.coli''. The DNA yield from the midi prep was 134μg as determined by spectrophotometry. This is the XylE that is going to be used for all further experiments.
-
* Dephosphorylation of pSB1C3
+
* Restriction digestion of midi prepped XylE by Xbal and PstI to prepare it for 3A assembly. (with J23101 promoter and PSB1C3 vector)
-
* Set up ligation of pSB1C3 and BOO14 over night
+
* Gel analysis of the restriction digestion mixture to isolate XylE gene
-
||
 
-
* Transformation of E. ''coli'' with ligation product pSB1C3-BOO14
+
Midi prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).
-
||
+
* I made an overnight culture of ''Bacillus''
-
* Analysis of colony PCR using gel electrophoresis
+
-
* ''Meeting with the supervisors''
+
-
||
+
[[Image:IC_Midi-prep_XylE_digestion_with_xbaI_and_PstI.jpg|thumb|center|400px|Midi prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).]]
-
*Repeat colony PCR with uncontaminated reagents
+
'''Tuesday, 17-Aug-2010'''
-
*Analysis of PCR product with gel electrophoresis
+
* Using the Gel Extraction Kit, we isolated the restriction enzyme cut XylE gene from the agarose gel band.
-
*Restriction digest with EcoRI and SpeI to confirm correct insert (BOO14)
+
* Gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine their ratios for 3A assembly ligation
-
*Gel electrophoresis to analyse restriction fragments
+
* 3A assemply of XylE, J23101 promoter and pSB1C3 vector.
 +
* Transformation of XL-Blue competent ''E.coli'' with the above construct.
-
||
+
[[Image:IC_XylE-J23101-pSB1C3_Clone_Gel2.JPG|thumb|center|400px|gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation]]
-
* Gel electrophoresis of the PCR product to confirm correct amplification
+
gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation
-
* Set up midi prep cultures of pSB1C3-BOO14
+
* I followed Chris's ''Bacillus'' transformation protocol to transform ''Bacillus'' with constitutive GFP and RFP DNA as well as a control without DNA
 +
{| style="width:800px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Successful Bacillus transformation!
 +
|-
 +
|align="center"|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/fFwYCh5wvfc?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/fFwYCh5wvfc?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html>
|}
|}
 +
'''Thursday, 19-Aug-2010'''
 +
* We run the XylE-GFP1 PCR reaction to construct the His-GFP-Flag and Linker-XylE-Spe construct.
-
====Monday, 17th-Aug-2010====
+
'''Friday, 20-Aug-2010'''
-
*We successfully purified BOO14 from the gel using the ''QIAquickÆ Gel Extraction Kit (250)'' but used 50µl of ddH2O instead of elusion buffer in the last step.  
+
The J23101 gene in a BioBrick vector containg RFP gene
-
*Then we determined the relative concentrations of BOO14 and pSB1C3 by gel electrophoresis and comparing the intensity of the bands with the 1kb ladder.
+
* We run a gel on XylE-GFP1 PCR reaction. Results: GFP was extended successfully, XylE extension FAILED (too much non-specific annealing)
-
*Having determined the rough concentration of our DNA we set up over night ligation of pSB1C3 with BOO14. We used two different ratios of pSB1C3 to BOO14: 0.5µl:3.5µl and 1µl:3µl. As the final concentration of the ligation product is very low, we will transform E. ''coli'' to be able to analyse bigger quantities of the vector at a later point.
+
* Catechol assay on 2hrs bench ligation of promoter, XylE and vector failed.
 +
* Two replica plates of overnight ligated J23101, XylE and pSB1C3 transformed ''E.coli'' (one for catechol assay)
 +
* The His-GFP-Flag DNA was gel purified
 +
* Transformation of XL-1Blue cells with J23101 in J62001 vector from the registry. One more attemp to construct a successful promoter-XylE ligation, since we believe that the strand annealed promoter was of bad quality
 +
|}
-
====Tuesday, 18th-Aug-2010====
+
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 8
 +
|-
 +
|
 +
<html>
 +
<table width="850px" border="0">
 +
  <tr>
 +
    <td style="background-color:#FFFF66;text-align:center;color:#555555;">
 +
      <b>Week 8</b>
 +
    </td>
 +
<td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Monday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Tuesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Wednesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Thursday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Friday</b>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Morning</b>
 +
    </td>
-
*E. ''coli'' was transformed with the pSB1C3-BOO14 ligation using chemical competence and heat shock. We prepared plates with our transformants to be incubated overnight.
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
*We reorganized and updated our Lab-Page on the Wiki, including new tables, upload of pictures and result as well as user-interface optimisation.
+
<ul>
 +
<li>PCR extention of His-GFP-flag round 2</li>
 +
</ul>
 +
</td>
-
====Wednesday, 19th-Aug-2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>midi prep registry obtained J23101</li>
 +
<li>gel purification of XbaI-His-GFP-flag-TEVs</li>
 +
</ul>
 +
</td>
-
*The plates with pSB1C3-BOO14 transformants had colonies growing on it.
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
*Some of these colonies were tested by colony PCR to confirm that BOO14 was in the vector.
+
<ul>
-
*Gel electrophoresis of the PCR product indicated that our ligation and transformation were successful, however due to contamination, as demonstrated by our negative control, we will have to repeat the colony PCR on Thursday. We set up an additional PCR to test the individual components of our PCR reaction to find out which one is contaminated with DNA.
+
<li>gel analysis of overnight ligation and gel separation</li>
-
*We also set up cultures of pSB1C3-BOO14 for mini preps tomorrow.
+
<li>gel analysis of linker-XylE-SpeI PCR reaction </li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>make replica plate and catechol assay plate </li>
 +
</ul>
 +
</td>
-
====Thursday, 20th-Aug-2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>catechol assay</li>
 +
</ul>
 +
</td>
 +
</tr>
-
*Analysis of the PCR components indicated that the Barns buffer was contaminated with DNA.
+
  <tr>
-
*We prepared mini preps of pSB1C3-BOO14 from the cultures set up yesterday.
+
<td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Afternoon</b>
-
*We then repeated the colony PCR and analysed the product with gel electrophoresis. We observed a band at 100bp, which indicates that the ligation was successful, and that BOO14 is in pSB1C3.
+
    </td>
-
*We tried to confirm this with a restriction digest of the mini prepped plasmid with EcoRI and SpeI, however, analysis with gel electrophoresis showed there was only a very faint band on the gel at 100bp maybe because the gel had been run too long.
+
 
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Gel purification of linker-XylE-Spe PCR construct</li>
 +
<li>Gel analysis of His-GFP-flag round 2 PCR rection and then gel separation of DNA </li>
 +
</ul>
 +
</td>
-
====Friday, 21st-Aug-2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
'
+
<ul>
-
*We repeated the gel electrophoresis but reduced the time it ran for to 10 minutes as BOO14 is very short. However we still only observed a very faint band at 100bp, so we can't be completely sure that the ligation was successful.
+
<li>restriction digestion, gel analysis and gel purification of registry obtained J23101 </li>
-
*Therefore we carried out another restriction digest with AseI (which cuts within B0014) and NcoI (which cuts within pSB1C3).
+
<li>PCR extension of linker-XylE-SpeI</li>
-
*We also performed a PCR to amplify LytC cell wall binding domain (CWB) from the Bacillus genome.
+
<li>ligation of pSB1C3, J23101 and XylE (overnight)</li>
-
*Analysis of the PCR product with gel electrophoresis showed that lytC had not been amlified properly. There we will do a series of different PCR reaction tomorrow to determine the optimal temperatures.
+
</ul>
 +
</td>
-
====Sunday, 22nd Aug 2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>gel purification of overnight ligation</li>
 +
<li>transformation of ''E.coli'' with overnight ligation product and selection by plating</li>
 +
<li>gel purification of TEVs-linker-XylE-SpeI construct </li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>PCR construction of fusion protein</li>
 +
</ul>
 +
</td>
-
*Analysis of the restriction fragments from yesterdays restriction digest with gel electrophoresis showed that:
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
#The ligation definitely worked! The correct fragment sizes were observed on the gel.
+
<ul>
-
#The primers in the PCR may have annealed nonspecifically, because the PCR product did not resolve properly in the gel.
+
<li>Gel analysis and gel purification of fusion XylE protein</li>
 +
</ul>
 +
</td>
 +
</tr>
 +
</table>
 +
</html>
-
*To prepare the ligated plasmid pSB1C3-BOO14 for midi prep tomorrow, overnight cultures were set up using 100ml LB (containing chloramphenicol), which was innoculated with cells from the original replica plates.
+
'''Monday, 23-Aug'''
-
*A second PCR was set up with a gradient of increasing annealing temperatures, which would hopefully ensure specific binding of primers. (We used taq in this case, just to see which temperature gave us the best result)
+
* Set up overnight cultures for midi prep
-
*The gel of the PCR products showed that all 3 PCRs were successful. So we can now use Pfu in the next PCR to obtain the LytC cell wall binding domain.
+
* Gel separation of linker-XylE-Spe DNA <font color = red>'''FAILED'''</font>
 +
* PCR reaction for extension of His-GFP-Flag
 +
* Catechol assay on ''E.coli'' transformed with overnight ligated J23101, XylE and pSB1C3. <font color = red>'''FAILED'''</font>
 +
'''Tuesday, 24th-Aug'''
 +
* Midi prepped registry obtained J23101. A yield of 130ng/μl of promoter was obtained. The promoter is in a BioBrick vector called J62001. The promoter is upstream of RFP gene.
 +
*the vector carrying the promoter was digested with SpeI and PstI, while XylE gene was digested with XbaI and PstI.
 +
* The promoter and the XylE gene were gel purified.
 +
* A reaction between the promoter(still on vector) and XylE was set on for overnight ligation
 +
* PCR purification of GFP2 -> GFP construct ready for full fusion protein.
 +
* Gel purification of XylE lost DNA along the way. Thus PCR XylE1 with gradient for temperature scanning (taq): PCR round 1 included 62°C and only rev primer to create pool of successful extensions with with rev primer (60-62°C). PCR round 2 with Fwd primer and temperature scale (68-68°C, 72-74°C).
-
===Week 8===
+
'''Wednesday, 25th-Aug'''
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
+
Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appalling (explanation for Nick: really bad) results, 2nd gel run was successful.
 +
* Performed a ligation reaction between the vector containing J23101, and XylE (one on bench and one overnight one).
 +
* Transformation of the new plasmid into competent ''E.coli''. Successfully transformed colonies can be selected for by loss of RFP expression.
 +
* XylE-1 PCR with temperature cascade. Gel analysis and purification.
-
|-
+
'''Thursday, 26th-Aug'''
-
  ! Week 8 !! Monday, 23rd-Aug-2010 !! Tuesday, 24th-Aug-2010!! Wednesday, 25th-Aug-2010 !! Thursday, 26th-Aug-2010 !! Friday, 27th-Aug-2010
+
-
|-
+
-
  | '''MORNING''' ||
+
-
* Midi prep of pSB1C3-BOO14
+
* White colonies from the promoter-XylE transformed ''E.coli'' were picked and transferred to new amp plates. One is the replica plate and the other is the catechol assay plate.
-
* PCR of lytC using Pfu polymerase
+
* XylE-1, two rounds of PCR/purification were run to obtain a sufficiently clear band. An additional PCR run for XylE-1 was discarded afterwards.
-
* DpnI restriction digest of PCR product
+
-
||
+
'''Friday, 27th-Aug'''
-
* Restriction digest of lytC with XbaI
+
* Catechol assay performed on promoter-XylE transformed ''E.coli''. <font color = green>'''SUCCESSFULL'''</font>
 +
[[Image:IC_Catechol_Assay_before.jpg|thumb|left|Plate before adding catechol assay]] [[Image:IC_Catechol_assay_after_(27-8).jpg|thumb|center|After addition of catechol colonies turn yellow-orange in seconds!!]]
 +
* XylE-2 PCR and gel-purification cycles (2x) to obtain clear band. XylE-2 is now ready for assembly of the GFP-XylE fusion protein.
-
||
+
'''Saturday, 28th-Aug'''
-
* Midi prep of pSB1C3-BOO14
+
* Preparing the annealing step between the GFP-2 and XylE-2 constructs, we discovered sequence dissimilarities in the TEV-cleavable regions which we planned to use for the annealing step. Nevertheless a PCR was run with appropriate conditions (allowing for a minimal amount of unspecific annealing).
-
* Gel electrophoresis to confirm correct midi prep product
+
-
   
+
-
||
+
-
* Restriction digest of pSB1C3-BOO14 with XbaI and SpeI
+
'''Sunday, 29th-Aug'''
-
* Gel electrophoresis to measurement of relative concentrations of pSB1C3 and lytC after restriction digest
+
-
* Dephosphorylation of pSB1C3
+
-
* Ligation of pSB1C3 with lytC
+
-
* PCR of promoter pVeg out of vector using taq
+
-
||
+
* Gel analysis of the attempted annealing reaction of GFP-2 XylE-2 showed unsufficiently clear bands for gel-purification. A new reaction is being prepared: 10 rounds of annealing PCR, followed by addition of primers (5' primer for GFP-2 and 3' primer for XylE-2) in order to introduce an amplification step in the reaction. --- Following Kirstin's advice, we are discarding this reaction and wait for the arrival of new primers for XylE-2 (5' + TEV) and GFP-2 (3' +TEV).
-
* Screen plates for successful transformation
+
|}
-
* Gel electrophoresis of pVeg PCR sample 2 and PCR purified sample 1.
+
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 9
|-
|-
-
  | '''AFTERNOON''' ||  
+
|
 +
<html>
 +
<table width="850px" border="0">
 +
  <tr>
 +
    <td style="background-color:#FFFF66;text-align:center;color:#555555;">
 +
      <b>Week 9</b>
 +
    </td>
 +
<td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Monday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Tuesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Wednesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Thursday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Friday</b>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Morning</b>
 +
    </td>
-
* Gel electrophoresis of lytC
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* Gel purification of lytC
+
<ul>
-
* Midi prep of pSB1C3-BOO14
+
<li>mini prep of promoter-XylE transformed ''E.coli''</li>
-
* Overnight digestion of lytC with SpeI
+
<li>design of reverse primer for GFP to add the corrected TEV sequence to the construct.</li>
 +
</ul>
 +
</td>
-
  ||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>midi prep of promoter-XylE transformed ''E.coli''</li>
 +
<li>restriction digestion of pSB1C3+terminator vector DNA</li>
 +
<li>restriction digestion of promoter-XylE DNA </li>
 +
</ul>
 +
</td>
-
* Set up new pSC1C3-BOO14 culture for midi prep
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Gel purification kit of cut promoter-XylE DNA</li>
 +
<li>Preparation of M9 and LB medium for assay </li>
 +
<li>Gel analysis of of insert and vector DNA</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>find optimun wavelength for catechol assays</li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>find optimun cell density and catechol concentration for assay</li>
 +
<li>Reverse primer for GFP with modified TEV arrived - primer dilution</li>
 +
</ul>
 +
</td>
 +
</tr>
-
* Restriction digest with XbaI and SpeI  to confirm BOO14 in pSB1C3
+
  <tr>
-
* Gel purification of restriction product
+
<td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Afternoon</b>
-
* Gel electrophoresis to analyse restriction product
+
    </td>
 +
 
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>gel analysis of mini prepped promoter-XylE</li>
 +
<li>cross-check for primer design (GFP-TEV-2) and ordering</li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>PCR purification of cut pSB1C3+terminator vector DNA </li>
 +
<li>Gel analysis of cut promoter-XylE DNA</li>
 +
</ul>
 +
</td>
-
* Ligation for transformation
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* PCR of LytC using Pfu for high fidelity
+
<ul>
 +
<li>dephosphorylation of the vector+terminator</li>
 +
<li>ligation reaction between promoter-XylE insert and pSB1C3+terminator vector</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>GFP 2 PCR with new reverse primer (adopted TEV sequence)</li>
 +
</ul>
 +
</td>
 +
</tr>
 +
</table>
 +
</html>
-
* Restriction digest of pVeg with EcoRI and XbaI
 
-
|}
+
'''Monday, 30th-Aug'''
 +
* Mini prep of 4 x J23101-XylE taken from 4 different colonies (8 14 24 27).
 +
* The gel analysis showed successful vector uptake.
 +
* Set up an overnight culture for midi using colony 24 (gel analysis showed similar results colony 24 was picked randomly.)
 +
* A new primer was designed in order to add a corrected TEV-protease-cleavable sequence to the His-GFP-Flag construct. This was controlled and ordered.
-
====Monday, 23rd Aug 2010====
+
'''Tuesday, 31st-Aug'''
 +
* Midi prep of colony 24 for XylE-J23101 the final concentration was ~100ng/μl which wasnt so great but Chris says the protocol produces very poor yields.
 +
* We are performing the next building step of our vector. PSB1C3 containing terminator B0014 was cut with EcoRI and XbaI. The insert was cut with EcoRI and SpeI and both were incubated for 1.5hrs. Wolf is now running a gel to purify out the insert via gel purification and perform a PCR purification on the vector.
 +
*Advisors have decided it's best not to use Jeremy's tagged XylE due to the 93% difference. Kirsten will be tagging the registry XylE and we shall purify and assay with that instead.
 +
*We shall see purification expert Kieran tomorrow and talk through the process. Chris will also talk us through our characterization of XylE experiments- we will use the robot after it's been programmed but until then we can use the plate-reader.
-
*We successsfully performed PCR using Pfu polymerase to amplify the LytC CWB domain.
+
'''Thursday, 2nd-Sept'''
-
*The PCR product was then digested with DpnI to remove template DNA and then gel purified.
+
[[Image:IC_200-600nm_spectr.jpg|thumb||center|300px|Spectra of XylE transformed E.coli after addition of catechol assay. The '''broad peak around 380nm wavelength''' arises is due to the presence of the product of the enzymatic reaction involving pyrocatechol and XylE enzyme. This peak if absent if a culture of XylE transformed cells are measured without the addition of catechol]]
-
*pSB1C3-BOO14 was midi prepped, and the final DNA concentration was measured to be 120ng/µl.
+
*Spectrophotometry experiments with XylE transformed ''E.coli'' in LB medium (M9 culture was contaminated) reveiled the followings: On catechol assay of the trasformed cells, the '''positive yellow output can be quantitively measured by a broad peak at 380nm.'''
-
*Overnight digestion of lytC with SpeI was set up because restriction will be inefficient on the PCR product. XbaI will be added to the mixture in the morning making the final volume of the digestion 30µl.
+
*Transformation of competent ''E.coli'' cells with promoter-XylE-terminator pSB1C3vector.
-
====Tuesday, 24th-Aug-2010====
+
'''Friday, 3rd-Sept'''
-
*XbaI was added to the restriction digest of lytC. PCR purification was performed to isolate the CWB.
+
*experiment to determine concentrations of catechol and cell density for assays
-
*Due to problems with our midi-prep of pSB1C3, we only digested the two components but then had to set up another culture of B. ''subtilis'' for another round of midi prep.
+
*The new GFP + TEV primer arrived, was diluted and used to set up the appropriate PCR.
-
====Wednesday, 25th-Aug-2010====
 
-
*We performed another midi prep using the culture of pSB1C3-BOO14 set up yesterday.  
+
[[Image:IC_Assay_3_sept.jpg|thumb|center|400px|Catechol assay on XylE-trasformed cells in a 96-well plate (A to H decreasing cell concentration, 1-10 decreasing catechol concentration, column 11 and 12 negative and control)]]
-
*After a restriction digest of the plasmid with XbaI and SpeI and consequent gel purification the product was analysed with gel electrophoresis and the plasmid was found to contain BOO14 so that this midi prep can be used for the following steps.
+
|}
-
 
+
-
====Thursday, 26th-Aug-2010====
+
-
 
+
-
*We performed a restriction digest of pSB1C3-BOO14 with XbaI and SpeI.
+
-
*We then determined the relative concentrations of lytC and pSB1C3 to prepare ligation. We found that pSB1C3 had a about 4 time higher concentration than lytC.
+
-
*We set up a 10µl dephosphorylation reaction with 2µl of pSB1C3 and incubated for 10min followed by heat deactivation of the T4 alkaline phosphatase.
+
-
*2.5µl of the dephosphorylation reaction were used together with 2µl of lytC for a 10µl ligation reaction. This was split into a 5µl overnight ligation and a 5µl bench ligation reaction.
+
-
*After 2 hours incubation at room temperature we transformed E. ''coli'' with the ligated vector and later plated it.
+
-
*We set up a test PCR using taq polymerase to determine the best protocol to get the promoter pVeg out the current vector pSB1AK3.
+
-
*Gel electrophoresis of the PCR products suggested that the following protocol was most suitable:
+
-
# 5 cycles at 60∞C
+
-
# 25 cycles at 66∞C
+
-
*We set up another PCR reaction using Pfu for to obtain two identical samples of pVeg with high fidelity
+
-
*Sample 1 was later PCR purified by Kirill and Kyascha (thanks guys!)
+
-
 
+
-
====Friday, 27th-Aug-2010====
+
-
 
+
-
*Both samples of pVEG were successfully puified and can both be used in the following cloning steps
+
-
*Our transformation was unsuccessful and no background was observed either. We thus used our overnight ligation to transform E. ''coli'' again.
+
-
*We decided to digest the promoter with EcoRI and SpeI rather than EcoRI and XbaI as originally planned. Digestion with SpeI was set up overnight but the digestion volume was accidentially made up to a total of 30µl rather than 28.5µl allowing for EcoRI to be added the next day.
+
-
 
+
-
====Saturday, 28th-Aug-2010====
+
-
 
+
-
*Transformation of E. ''coli'' using our overnight digestion was unsuccessful which means we have to restart construction of our lytC vector.
+
-
*EcoRI was added to the pVEG digestion and both samples were incubated for 1 hour
+
-
*5µl of sample 1, which is to be PCR purified later, were loaded on a gel and 30µl (all) of sample 2, which is to be gel purified. The electrophoresis confirmed that pVEG had been digested and the bands of smaple 2 (two lanes in total) were cut out of the gel for gel purification (0.44g).
+
-
 
+
-
 
+
-
 
+
-
===Week 9===
+
-
 
+
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
+
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 10
|-
|-
-
  ! Week 9 !! Monday, 30th-Aug-2010 !! Tuesday, 31st-Aug-2010!! Wednesday, 1sth-Sep-2010 !! Thursday, 2nd-Sep-2010 !! Friday, 3rd-Sep-2010
+
|
-
|-
+
-
  | '''MORNING''' ||
+
-
*Gel Purify pVEG (digested E+S) sample 2
+
<html>
 +
<table width="850px" border="0">
 +
  <tr>
 +
    <td style="background-color:#FFFF66;text-align:center;color:#555555;">
 +
      <b>Week 10</b>
 +
    </td>
 +
<td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Monday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Tuesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Wednesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Thursday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Friday</b>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Morning</b>
 +
    </td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>PCR extension of PVeg promoter </li>
 +
</ul>
 +
</td>
-
*Digestion of pSB1C3 EcoRI + SpeI
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
*Gel electrophoresis of digestion product
+
<ul>
 +
<li>Gel purification of extended PVeg promoter</li>
 +
<li>Annealing PCR for GFP-XylE fusion protein</li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>vector with insert tranformed into ''E.coli'' and plate to select for successful transformation</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>prepare overnight cultures of pVeg-RBS transformed ''E.coli'' </li>
 +
<li>Gel-analysis of amplification-PCR for XylE-2</li>
 +
</ul>
 +
</td>
-
*Preparation of LB agar
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>midi prep of PVeg-RBS vector</li>
 +
</ul>
 +
</td>
 +
</tr>
-
||
+
  <tr>
 +
<td style="background-color:#FFCC66;width:100px;text-align:top;color:#555555;"><b>Afternoon</b>
 +
    </td>
 +
 
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Gel analysis and purification of the GFP-TEV construct</li>
 +
</ul>
 +
</td>
-
*Measuring DNA concentration in 5 midi-preps
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
*Update Wiki
+
<ul>
 +
<li>restriction digestion and gel purification of thr extended pVeg promoter</li>
 +
<li>overnight ligation of the cut pVeg promoter in a vector </li>
 +
<li>Gel analysis of GFP-XylE annealing PCR - unsuccessful reaction</li>
 +
</ul>
 +
</td>
-
||
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Amplification PCR for XylE-2</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Gel-purification of amplification-PCR for XylE-2</li>
 +
</ul>
 +
</td>
-
*Update Wiki
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
*Measure midi prep concentrations in the Biochem building
+
<ul>
-
*Check for successful transformations
+
</td>
-
*If we have transformants:
+
</tr>
-
# Make a replica plate
+
</table>
-
# Perform a colony PCR
+
</html>
-
|-
+
'''Monday, 6th-Sept'''
-
  | '''AFTERNOON''' ||
+
* PCR extension of pVeg promoter: EcoRI---pVeg---RBS-SpeI
 +
* I performed a catechol assay on the picked transformed colonies to deduce which ones were successfully transformed with the insert plus vector. 1-5 7 and 10 failed to turn yellow (1-5 were background controls)leaving 8 yellow colonies.
 +
* I had to perform a colony PCR on two selected colonies 8 and 14 to check the correct insert+terminator was present.
 +
* Colony 8 when run on a gel analysis showed the correct size 924 XylE + 97 J23101 + 35 B0014.
 +
* This was set up as an overnight culture.
 +
* Gel analysis of the GFP-TEV construct showed satisfactory bands.
-
||
+
'''Tuesday 7th Sept'''
 +
* Mini prep of the overnight Colony 8 culture (by Wolf)
 +
* Midi prep of overnight culture
 +
* Gel purification of PCR product EcoRI--pVeg--RBS--SpeI
 +
* Overnight restriction digestion of EcoRI--pVeg--RBS--SpeI with SpeI
 +
* Started data analysis of plate assay
 +
* Annealing PCR reaction included 2 samples without and one with additional primers (XbaI-His-GFP Fwd, SpeI-XylE Rev). While the primer-including reaction did not show any clearly identifiable bands, the others showed clear, if very weak bands at 800 and 1000bp, which represent the GFP and XylE constructs respectively. No band was identifiable at the 1.7 kb range, which would have indicated a successful annealing reaction. However, problems with the lense of the gel-analyser were only discovered later to have severely reduced the band brightness. Potentially a PCR-reaction with appropriate primers to amplify an annealed product(XbaI-His-GFP Fwd, SpeI-XylE Rev), could have been successful. However the reaction mixture was disposed of before this became clear.
-
*Gel purification of pSB1C3
+
'''Wednesday, 8th Sep'''
-
*Gel electrophoresis to determine relative concentrations of insert and vector
+
* A second PCR extension of pVeg promoter to introduce the RBS and cut sites. It was also gel purified and stored in gel lumbs in the freezer. (maybe needed later so keep in mind).
-
*Set up of overnight ligation
+
* Overnight restriction digestion completed. Then, it was run on the gel to check if it worked and then gel purified again.
 +
* Vector PSBI-C3 was digested to remove the terminator and make it sticky for the insert (pVeg-RBS). Then it was run on the gel to check if restriction has worked, but the gel didn't run far enough to determine easily between undigested and digested vector.
 +
* For further annealing reactions for GFP-XylE constructs additional XylE(2) template was required -> amplification PCR for XylE(2).
-
||
+
'''Thursday, 9th Sep'''
 +
* Gel with digested and undigested vector PSBI-C3 was run and then the digested vector was purified.
 +
* Gel was run to determine the DNA concentration ratio for the ligation of PSBI-C3 and pVeg-RBS.
 +
* Vector PSBI-C3 was dephosphorylated.
 +
* Ligation of PSBI-C3 and pVeg-RBS has been set up overnight.
 +
* Gel-analysis and gel-purification of the XylE(2) amplification PCR product.
 +
* The transformation of ''E.Coli'' with pVeg-RBS in PSBI-C3 and PSBI-C3 by itself (to check see how successful dephosphoryaltion of PSBI-C3 was and estimate the percentage of bacteria that contain the insert) was completed
-
*Meeting with supervisers
+
* Concentration of the midi prep of J23101-XylE-B0014 was determined to be ~600ng/μl (using new protocol)
 +
* Kyasha kindly digested my midi with EcoRI and SpeI and performed a gel analysis. The results show a potential additional plasmid contaminating my midi however the concentration of DNA was extremely high. NB Chris said that it could be sheared DNA from a midi prep step.
-
||
+
* The midi prepped plasmid was transformed into testing ''E.coli'' strain TOP10.
-
*try to measure midi prep concentration
+
'''Friday, 10 sep'''
 +
The transformation was a SUCCESS. 2x replica plates were made plate 1# 1-6 plate #2 6-11; colony 6 and colony 9 of plates 1 and 2 respectively were transfered into a 5ml liquid culture + 5μl CmR. These will later be turned into glycerol stocks. After the replica plates have grown up mini preps on a number of colonies shall be performed - this hopefully will eliminate the contaminating plasmid DNA. This will be followed by a midi prep.
-
||
+
'''Saturday, 11 Sep'''
-
 
+
-
*Run gel electrophoresis  to analyze colony PCR results
+
 +
The transformation of ''E.Coli'' with PSB1-C3 with insert did not work :(
|}
|}
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 11
 +
|-
 +
|
 +
<html>
 +
<table width="850px" border="0">
 +
  <tr>
 +
    <td style="background-color:#FFFF66;text-align:center;color:#555555;">
 +
      <b>Week 11</b>
 +
    </td>
 +
<td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Monday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Tuesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Wednesday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Thursday</b>
 +
    </td>
 +
    <td style="background-color:#FFFF99;text-align:center;color:#555555;"><b>Friday</b>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Morning</b>
 +
    </td>
-
====Monday, 30th-Aug-2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>Gel-analysis of amplification-PCR for XylE-2</li>
 +
</ul>
 +
</td>
-
*Both sample of pVeg which had been digested with EcoRi and SpeI were purified: Sample 1 was PCR purified (by another team) and sample 2 gel purified. Two methods of purification were used because pVEg is so small that either method posed the risk of loosing our product.
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
*Gel electrophoresis later confirmed that both samples were purified successfully.
+
<ul>
-
*We also set up a PCR of lytC for blunt ended ligation as an alternative to our previous cloning strategy.
+
<li>midi prep of pVeg-RBS vector</li>
 +
</ul>
 +
</td>
-
====Tuesday, 31st-Aug-2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>In preparation of ''catechol cell-death assay'' tranformation of Top10 cells with CMR plasmid (psb1C3)</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
</ul>
 +
</td>
 +
</tr>
-
'''Plan:'''
+
  <tr>
-
* Digestion of pSB1C3 EcoRI + SpeI
+
<td style="background-color:#FFCC66;width:100px;text-align:center;color:#555555;"><b>Afternoon</b>
-
* Gel electrophoresis of digestion product
+
    </td>
-
* Gel purification of pSB1C3
+
 
-
* Gel electrophoresis to determine concentrations of insert and vector
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* Dephosphorylation of pSB1C3
+
<ul>
-
* Set up of overnight ligation
+
<li>Gel-purification of amplification-PCR for XylE-2</li>
 +
</ul>
 +
</td>
-
'''Report:'''
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* Restriction digest of pSB1C3-BOO14 was performed with EcoRI and SpeI to remove the terminator and later allow ligation with pVeg.
+
<ul>
-
* Gel electrophoresis was used to confirmed that digestion of pSB1C3-BOO14 with EcoRI and SpeI was successful.
+
</ul>
-
* The digested vector was then gel purified and the relative concentrations of the digested pSB1C3 and pEVG were determined. Our samples contained about 30 times as much vector as promoter and the vector is about 20 times larger than the insert.
+
</td>
-
* therefore we decided to ligate overnight in a ration of 1.5 volumes of pVEG to 1 volume of dephosphorylated vector.
+
-
* LytC was digested overnight with SpeI.
+
-
====Wednesday, 1st-Sep-2010====
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>plating and overnight incubation of transformed top10 CMR</li>
 +
</ul>
 +
</td>
 +
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
 +
<ul>
 +
<li>overnight cultures for catechol cell death assay: Top10 cells XylE/CMR </li>
 +
</ul>
 +
</td>
-
* We performed another restriction digest of pSB1C3-BOO14 using XbaI and SpeI.
+
<td style="background-color:#eeeeee;height:100px;width:200px;text-align:left;color:#555555;">
-
* XbaI was added to the restriction digestion of lytC.
+
<ul>
-
* Correct digestion of lytC and pSB1C3-BOO14 was confirmed using gel electrophoresis and pSb1C3 as well as lytC were then gel purified. All planned steps worked and overnight ligation of pSB1C3 and lytC were set up.
+
</td>
-
* We also prepared some LB agar today and had a meeting with the supervisors.
+
</tr>
 +
</table>
 +
</html>
 +
 +
'''Monday 13th september'''
 +
Overnights weren't set up on sunday so they were made up alongside some assay cultures. J23101-XylE-B0014 colonies 8 and 10 of the replica plate #2 were picked. Chris also provided a replica plate containing 3k3 vector colonies. This was over a year old and he was unsure whether it was the correct plasmid or if the cells would grow up. I picked all available colonies 102 150 151 and 260 of kanamycin resistance. Lastly for assays 2x LB 2xM9 cultures were made 5ml +5ul antibiotic.
-
====Thursday, 2nd-Sep-2010====
+
'''Tues 14th September'''
 +
[[Image:CS1.JPG|thumb|right|300px|test confirming that yellow product of XylE enzmymatic reaction is leaks back from the cytosol into the solution.1C and 2C is the XylE producing cells after centrifuging and redissolving of the pellet and 1S and 2S is the supernatant after centrifuging]]
 +
*assay on plate reader of:
 +
**0-0.75 initial O.D. of transformed ''E.coli''
 +
**0-1mM initial catechol concentration
 +
The assay was carried out with ''E.coli'', top ten spcies, transformed with J23101-XylE-B0014 in pSB1C3 vector.The overnight culture wastransfered in new medium this morning for 4 hrs before assaying. LB medium was used for dilutions and blank. Catechol was diluted in ddH2O.
 +
[[Image:Catechol assay test 2.xls|Data analysis of the assay]]
 +
*Mini preps of XylE 8,10 colonies and 3K3;102 151 260 colonies. Analytical digests were performed using E+S on XylE, E+S AccI and HindIII+E for 3K3 vector respectively. The band patterns correctly identified the vector to be 3k3. Colony 10 of XylE and Colony 260 of the 3k3 vector were set up direct into 100ml LB and so will require til 12pm for midi prepping on weds. Nick performed a second plate reader assay to determine the minimal concentration of Catechol to use for assays. I took 990ul M9 culture containing colony 10 and 10ul catechol was added. This was incubated for 10 mins and then spun down. The supernatant was removed into a cuvette and the cells resuspended in M9 salts. The ODs were then read on the spectrophotometer at 380 and 600nm. It was found that control cuvette of M9 salts had a miniscule reading. M9 + cells ~0.007. Supernatant + cells were ~ 2.5 therefore we could deduce that the coloured catechol breakdown product is exported out of the cells.
-
*We tried to measure the concentration of 5 midi preps Chris had made of linkers from synthesis. However the spectrophotometer caused great problem as it continuously, and even when operated by different people, produced wrong and inconsistent results as well as failing to remain callibrated.
+
Piotr:
-
*We therefore used gel electrophoresis to determine the sample concetrations (2 lanes each, 1µl and 5µl).
+
* Mini prep of 1 sample (6 samples lost due to mistake) of GFP-Xyle fusion protein and its digestion with Spe and Xba (gel to be run on the next day)
-
*However gel electrophoresis also yielded bad results so we will measure concentrations tomorrow using a different method.
+
* Preparing ''E.Coli'' colonies for the next day for mini prep to be redone
-
*We spend some time updating the Wiki today.
+
* PCR of 6 samples of GFP-Xyle fusion
-
====Friday, 3rd-Sep-2010====
+
'''Weds 15th September'''
-
*We finally measured the concentration of DNA in the midi-preps (of plasmids containing synthesis products). The concentrations varied between 78ng/µl and 230ng/µl and one will have to be repeated as the yield was too low.
+
* Midi preps of overnights.
-
*The transformation of the LytC-pSB1C3 was had produced many colonies. 20 colony PCRs were set up and a replica plate made.
+
* In preparation for assays determining the effect of catechol and/or breakdown product on cell viability we prepared Top10 cells, one strain containing pVeg-XylE-terminator and the other containing a CMR plasmid of similar size. As the XylE cells have already been prepared, only Top10 transformation with CMR-plasmid had to be carried out.
-
*Unfortunately gel electrophoresis suggested that the ligation was not successful, so another round of gel electrophoresis will be performed on monday to confirm these results.
+
 +
'''Thurs 16th September'''
 +
*Top10 CMR transformation was successful. Four overnight cultures of each Top10 XylE and Top10 CMR were set off with either M9 or LB medium respectively.
 +
*Piotr: Did mini preps from bacteria with blunt ended Xyle-GFP fusion and did diagnostic using both:
 +
- digest with XbaI and SpeI
-
===Week 10===
+
- PCR reactions with the following primers added: HIS-GFP, XylE-Xba and the other sample with HIS-GFP and GFP-flag
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
+
The PCR reactions were not very conclusive on the gels but digests allowed to determine that colonies 1->5 seem to have the right sizes of DNA in them and on Friday they will be prepared to be sent off for sequencing
-
|-
+
* Continuation of the midi from overnight step. A gel analysis was run which showed the correct bands were present. A gel purification of the protein was then performed and the bands excised.
-
  ! Week 10 !! Monday, 6th-Sep-2010 !! Tuesday, 7th-Sep-2010!! Wednesday, 8th-Sep-2010 !! Thursday, 9th-Sep-2010 !! Friday, 10th-Sep-2010
+
-
|-
+
-
  | '''MORNING''' ||
+
-
*Repeat gel electrophoresis of 7th colony PCR
+
'''Friday 17th September'''
-
*20 further colony PCRs from the LytC transformants.
+
*Catechol assay of transformed ''E.coli'' Top ten(J23101-XylE-terminator) in M9 medium + data analysis
 +
**[[Image:M9 catechol concentrations assay alternative.xls|Data analysis]]
 +
**[[Image:Catechol assay in M9.xls| alternative]]
-
||
+
The gel purifications of 3k3 vector and XylE were used for ligation.
 +
3K3 was dephosphorylated and then ligated in a ratio 5:1 with the insert. This was left overnight and Chris tried to transform with it on sunday.. ligation and transformation failed :-(
 +
|}
-
*Minipreps from overnight cultures.  
+
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 12
 +
|-
 +
|
 +
'''Monday 20th Sept'''
 +
Performed ligation again. Errors in the sequencing we received back regarding J23101-XylE-B0014.
 +
One was a log error (in XylE) phew. The other is in PSB1C3 out of the scar site and should be OK.
 +
Chris is currently assembling the pVeg promoter+ RBS in a vector. Once he has finished this, I will combine XylE-GFP and XylE with this promoter for comparison characterization with J23101 (in ''E.coli'' and ''Bacillus'')
-
||
+
'''Tues 21st Sept'''
 +
* Ligation to be transformed in a bit. (Issue with the vector not being cut efficiently=failed ligations?)
 +
* I am cutting the original XylE midi as well as PSB1C3 containing B0014 and ligating them today (hopefully) the vector is purified, waiting on the gel run and then extraction of XylE insert. A ligation ratio must be determined from a gel analysis then dephosphorylation of the vector and then ligation. This will produce a promoter-less XylE+terminator vector which will be readily available to switch in the desired promoter (E+S cut)
-
*Amplification of pVeg using PCR from pSB1AK3
+
* Florian is performing a mini prep on pVeg promoter which if it is correctly identified in the gel i can use to ligate to my XylE + PSB1C3-B0014.
-
*Gel electrophoresis to confirm correct amplification of pVeg
+
* Piotr is PCRing the reverse version of XylE that will be under the control of inducible promoter LacI (not delivered from synthesis yet) that will become a testing construct.
-
*Excision and gel purification of pVeg
+
-
||
+
'''Wed 22nd Sept'''
 +
* Piotr's PCR has been successful for 3 samples (1. recommended annealing temp; 2. recommended + 2 degrees and 3. recommended + 4 degrees). Sample no1. has been run on the gel and gel purified. It has been cut overnight with SpeI and tommorrow in the morning XbaI will be added.
 +
* I (Maddie!) performed steps towards the ligation (XylE-3K3) again from scratch (inefficient cutting steps??) including digest, gel analysis and extraction.
 +
* I ligated PSBC3-B0014 and XylE
 +
* I picked successfully transformed colonies for a replica plate and set up overnight mini cultures of 1,2,3 ComC DE promoter FWD and 4,5,6 ComC promoter Rev
-
*XbaI was added to overnight digestion of pVeg with SpeI
+
'''Thurs 23rd Sept First day of AUTUMN!!'''
-
*Gel electrophoresis to analyse pVeg restrcition digest
+
*I determined the ligation ratio for XylE and 3k3 (1:3) performed dephosphorylation of vector then ligation, to be transformed along with the other ligation tomorrow hopefully.  
-
*Because pVeg was lost during the last steps PCR amlification of pVeg from PSB1AK3 was repeated.
+
*I did mini preps of the Comc DE promoters, performed a ES test digest and am currently waiting for gel results. I shall then set up overnight midis.
-
*PCR purification of pVeg
+
*Miniprep results were fabulous, Ive chosen colony 1 and colony 4 which are ComCDE promoters FWD and REV respectively. They are made up into overnight midi cultures for tomorrow.
-
   
+
-
||
+
-
* Mini prep using overnight cultures of pSB1C3-lytC
+
'''Fri 24th Sept'''
-
* Restriction digest with EcoRI and SpeI as well as XbaI and SpeI for all 23 mini preps of pSB1C3-lytC
+
*Midi preps of ComC DE promoters FWD and REV (colonies 1,4)
-
* Culture of K5 for midi prep failed: New cuture was set up
+
*to be followed by a digest. XbaI Pst for FWD (not known for the reverse currently) to insert XylE after it.
 +
*Ligation with XylE and PSB1C3-B0014 possibly.
 +
*Transformation of XylE-PSB1C3-B0014 and of XylEe-3k3 into ''E.coli'' later
 +
* Hi Nick, hope revision is going well. Love from Team XylE xox
 +
|}
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 13
|-
|-
-
  | '''AFTERNOON''' ||
+
|
 +
'''Monday 27th Sept'''
 +
* Successfull transformations: now have XylE-PSB1C3-B0014 (no promoter) and 3K3-J23101-XylE-B0014. No background plate colonies..many colonies for 3K3-XylE and a few for PSB1C3-XylE. Colonies have been replica plated (plus catechol assay plate for 3K3-XylE) and set up for mini prepping tomorrow. 
 +
* Sumo tagged XylE will be given to us by Kirsten at some point this week and we will have to purify it. This can then be used for in vitro testing and comparisons made with the linker-XylE protein.
 +
* pVeg and ComC DE promoter FWD can be added into the promoter-less construct after it has been midi prepped and digested accordingly. ComC DE promoter REV can be added onto the reverse XylE construct (PCR'd by Piotr) AFTER it has been put into PSB1C3 and is more manageable. Kirsten is taking care of the GFP-XylE fusion for now, including putting the pVeg promoter plus RBS infront of it (proving difficult.) We still need to receive LacI promoter to create the inducible test version of the fusion XylE-GFP protein.
-
*Set up different mini prep cultures
+
'''Tues 28th Sept'''
-
*Gel electrophoresis to analyse colony PCR
+
* Performed mini preps and then E+S / E+S AND AseI digests of colonies 1-7 3k3-XylE and 1-3 C3-B0014-XylE respectively. Gel runs showed them to be the correct thing :)))))) so, 3k3-XylE final vector is now left as 2-7 minis (catechol assay test showed colony 1 was background so DO NOT USE) in my DNA box (orange tubes). Colony 1 of the C3-B0014-XylE was selected for midi prepping and will be performed tomorrow.
-
||
+
'''Weds 29th Sept'''
 +
* Midi prep of XylE-GFP fusion protein along with C3-B0014-XylE. The C3-B0014-XylE midi will then be used in 2x cloning digests (E+S) to insert pVeg and ComC DE FWD promoters. These will be obtained and also digested E+S to get out of their current vectors a gel analysis and purification will be run of all 3 in parallel, followed by a ligation ratio gel analysis, dephosphorylation of vector then ligation.
 +
* Primers for J23101-XylE arrive today so they can be used to PCR out J23101-XylE (From my J23101-Xyl-E midi) with blunt ends and this will be ligated into the final Spec vector so that we can get some data with Bacillus.
 +
* J23101-XylE-B0014 XylE-3K3 and PSB1C3-B0014-XylE were all sent off for sequencing
-
*Restriction digest of pSB1C3-lytC mini preps with XbaI and SpeI
+
'''Thurs 30th Sept'''
-
*Gel electrophoresis to analyse restriction fragments
+
* I gel purified ComCDE FWD promoter, C3-XylE-B0014, pVeg vector and XylE-B0014. Did a gel analysis to get ligation ratio.
 +
* Set up midi culture of J23101-XylE-B0014 colony 8
 +
* Set up midi culture to repeat GFP-XylE midi prep
 +
* Set up 6x minis (plus replica plate) of final Spec colonies 1-6
 +
* PCR reaction using newly arrived primers to get blunt ended J23101-XylE.
-
||
+
'''Friday 1st Oct'''
-
 
+
*morning maddie, from earl's court! :)
-
*Restriction digest of pVeg with XbaI and SpeI
+
ahahahaha awesome. hey you :)
-
*Gel electrophoresis of restriction fragments for analysis
+
-
*Diagnostic digest of pVeg was repeated with SpeI overnight
+
-
 
+
-
||
+
-
 
+
-
*Restriction digest of lytC in blunt-ended ligation vector
+
-
*Set up of midi prep cultur from K5
+
-
*Set up 23 mini prep cultures of pSB1C3-lytC
+
-
*Test culture was set up to test new chloramphenicol aliquot
+
-
||
+
-
 
+
-
* Repeat of digest of pVeg with SpeI overnight (EcoRI will be added tomorrow)
+
 +
* Today I peformed ligations for ComCDE FWD promoter and PSB1C3-XylE-B0014 and also PSB1C3-pVeg and XylE-B0014 they will be left overnight and transformed by chris tmro thaaanks.
 +
* I ran the PCR Kirill performed yday to get out blunt ended J23101-XylE on a gel; there was no template DNA! so I've just set up the reaction again... hopefully i'll purify it later for ligation into the final Spec vector that kyasha has maaaaade :))) then we can test XylE in ''Bacillus''.
 +
* I'm currently performing Midi's of my J23101-XylE-B0014 (running low) and of GFP-XylE (3rd time!)
|}
|}
-
====Monday, 6th-Sep-2010====
+
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|
 +
|-
 +
|
 +
have a look :)
 +
it's from John Hoppkins wiki 2008. There are one or two good ones!
-
* We repeated the gel electrophoresis for the 7th colony PCR, because it showed a faint band around 1kb, indicating that lytC was present in pSB1C3.
+
Love: Before I heard the doctors tell The dangers of a kiss;
-
* Simultaneously we set up another 20 colony PCRs for colonies from the pSB1C3-lytC ligation/transformation and made a replica plate.
+
-
* We also set up mini prep cultures from (a) the blunt-ended ligation of lytC and a vector and (b) numbers 5, 7, 18 and 19 of pSB1C3-lytC from the original colony PCR.
+
-
* The second colony PCR products were analysed using gel electrophoresis, but we think the primers may not be annealing because the bands at 1kb were not observed.
+
-
====Tuesday, 7th-Sep-2010====
+
I had considered kissing you. The nearest thing to bliss.
-
* We used the overnight cultures to make mini preps of both blunt-ended ligation of lytC and pSB1C3-lytC.
+
But now I know biology and sit and sigh and moan;
-
* We then performed a restriction digest of the pSB1C3-lytC mini preps with XbaI and SpeI to confirm that:
+
-
#The LytC cell wall binding domain is in the plasmid
+
-
#The CWB is in the correct orientation
+
-
* The uncut and cut plasmids were analysed with gel electrophoresis however the result suggested that ligation had been unsuccessful or that the restriction digest had failed.
+
-
====Wednesday, 8th-Sep-2010====
+
six million mad bacteria and I thought we were alone!
-
* We used PCR to amplify pVEG  from pSB1AK3 again.
 
-
* Gel electrophoresis confirmed that PCR of pVeg was successful although a big, strong band appeared far above the expected length of pVEG as well.
 
-
* pVEG was cut out of the gel and purified.
 
-
* A restriction digest of pVeg with XbaI and SpeI was performed.
 
-
* When analysing the restriction fragments using gel electrophoresis it appeared to have failed ''(it turned out later that one of the settings of the UV-box had been changed so not enough light was detected)
 
-
* pVEG was then digested with SpeI ovenight
 
-
====Thursday, 9th-Sep-2010====
+
Biology is the science of moving tiny droplets of invisible liquids from one tube to another.
-
* XbaI was added to overnigh digestion of pVeg with SpeI.
 
-
* The XbaI and SpeI restriction digestion of pVeg was analysed using gel electrophoresis but it pVeg had been lost.
 
-
* Therefore the PCR amlification from pSB1AK3 was repeated.
 
-
* This was followed by PCR purification which was then used for an overnight digestion with SpeI. Unlike last time, the gel purification was not used because it decreased our yield too much.
 
-
* A restriction digest of lytC in the blunt-ended vector was performed.
 
-
* Subsequent analysis by gel electrophoresis showed a possible candidate from Kirsten's blunt ended ligations (K5).
 
-
* A midi prep culture for K5 was set up.
 
-
* 23 mini prep cultures from our ligation plate of pSB1C3-lytC were set up.
 
-
* A test culture was set up to check if a new aliquot of chloramphenicol works.
 
-
====Friday, 10th-Sep-2010====
+
The human body was designed by a civil engineer. Who else would run a toxic waste pipeline through a recreational area?
-
*pVeg was set up in a restriction digest with SpeI
 
-
*The overnight culture of K5 failed because the wrong antibiotic was used. A new culture was set up with the correct antibiotic.
 
-
*23 Mini preps of lytC-pSB1C3 ligation were made.
 
-
*A restriction digest of lytC-pSB1C3 was performed with EcoRI and SpeI as well as XbaI and SpeI for the next day.
 
-
*Another restriction digest of pVeg was set up using EcoRI and SpeI this time (EcoRI will be added on the next morning).
 
-
====Saturday, 11th-Sep-2010====
+
I have a hunch that the unknown sequences of DNA will decode into copyright notices and patent protections.
-
*EcoRI was added to the pVeg restriction digest.
 
-
*Gel electrophoresis of the restriction digest of the mini preps indicated that restriction did not work as expected, maybe because one of the enzymes did not cut as a result of a wrong insert. Another possibility is that lytC is in pSB1C3 in the wrong direction, thus disrupting the restriction sites.
 
-
*Gel electrophoresis was used to analyse the restriction fragments of pVeg, which indicated that pVeg had been cut correctly.
 
-
*pVEG was cut out of the gel to be gel purified for ligation with pSB1C3.
 
-
*The K5 midi prep was started and paused after step 13 of the protocol.
 
-
====Sunday, 12th-Sep-2010====
+
It has recently been discovered that research causes cancer in rats.
-
*Digested pVeg was gel purified and ligated with digested, dephosphorylated pSB1C3.
 
-
*Restriction digest of pSB1C3-lytC with EcoRI and SpeI as well as EcoRI only was performed.
 
-
* Gel electrophoresis showed that SpeI did not cut the vector, probably as a result of incorrect ligation.
 
-
*The midi prep of K5 was completed
 
-
* when measuring the concentration of the midi prepit turend out to be close to 0ng/ul.
 
-
*3 mini prep cultures for K5 were set up.
 
 +
A mouse is an animal that, if killed in sufficiently many and creative ways, will generate a PhD.
 +
|}
-
 
+
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
-
===Week 11===
+
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 14
-
 
+
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
+
-
 
+
|-
|-
-
  ! Week 11 !! Monday, 13th-Sep-2010 !! Tuesday, 14th-Sep-2010!! Wednesday, 15th-Sep-2010 !! Thursday, 16th-Sep-2010 !! Friday, 17th-Sep-2010
+
|
-
|-
+
-
  | '''MORNING''' ||
+
-
*3 Mini-Preps of K5
+
'''Monday 4th Oct'''
-
*Restriction digest of K5 and pSB1C3-BOO14 with XbaI and SpeI
+
Did a replica plate and catechol assay plate/ minis 2 6 7 9 12 16. (Successful pVeg-XylE transformation)
-
||
+
'''Tues 5th Oct'''
 +
4 16 colonies were background the rest turned yellow. Mini prepped 2 6 7 9 12 16. Made overnight assay cultures of 3k3-XylE and C3-xylE
-
*Meeting with Prof Fenwick and Dr Harrison from the SCI
+
'''Weds 6th Oct'''
 +
* Digested mini preps 2 6 7 9 12 16 with E+S will run on gel to determine if it's the correct plasmid, then will select a colony to midi prep tomorrow.
 +
* Will perform the same assays already performed by Nick with the 3k3-XylE constructs to compare results.
 +
* Just realised Im working with background colony 16!! disregard this after the gel run!
 +
* I'm currently keeping my undigested pVeg-XylE minis in Florian's DNA box (orange lids and labels)
-
||
 
-
*Screen plates for transformed colonies
+
'''Thursday 7th Oct'''
-
*Set up mini prep cultures of pSB1C3-lytC and pSB1C3-pVeg
+
* Labpartner, goodmorning and from greece!! :p
-
*Wiki meeting
+
-
||
+
* Niiiiiiiiiiiiiiick!!! you left the country! you were too ugly. good luck babe!
-
*Mini preps of pSB1C3-lytC (7) were made but the pSB1C3-pVEG (4) mini preps failed
+
* Midi prepped pVeg-XylE-B0014 in PSB1C3; will digest out the insert (E+S) and put it into 3K3(E+X). Then both the midi (in PSB1C3) and the ligation 3k3-pVeg-XylE-B0014 can be transformed into TOP10. J23101-XylE-B0014 in 3K3 Vector also needs to be put into TOP10 currently only the PSB1C3 version is in TOP10. We need the rest for comparison testing as TOP10 is the more widely used testing strain.
-
*New cultures for mini preps of pSB1C3-pVeg were set up
+
* Florian kindly digested pVeg-XylE-B0014-C3 with E+S
 +
* Ran PCR to get out blunt pVeg-XylE-B0014 using primers. Did 3 reactions at variations around 60 degrees.
-
||
+
'''Friday 8th Oct'''
-
 
+
* Ran gel analysis of my PCRs 1, 2 and 3* appear to have worked. Samples 1 and 3* show stronger bands and so should be used in the next ligation step into the SPEC vector (which kyasha will be doing) then transformation into ''Bacillus'' (meeee.)
-
*Mini preps of pSB1C3-pVeg were successfully made
+
* Chris is purifying digested pVeg-XylE-B0014-C3 to get the insert out and this will then be ran on a gel alongside cut (E+X) 3k3 to determine a ratio for ligation.
 +
* Meeting today at 4pm, no one is here!! it'll be me Florian, Ben and Piotr attending..yikes
 +
'''Sunday 10th of the 10th of the 2010!!!!! and im in LAB '''
 +
* I did ligations of pVeg-XylE into 3k3 and of Reverse-XylE into the digested C-Tev LacI vector.
 +
* I also set up assay cultures for monday 2x pVegXE 2x XE-C3 2x XE-3K3 and 2x GFP-XylE!!!
 +
|}
 +
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
 +
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Week 15
|-
|-
-
  | '''AFTERNOON''' ||
+
|
-
*Gel electrophoresis of restriction fragments
+
'''Monday 11th Oct'''
-
*Gel purification of digested K5 and pSB1C3
+
*No Nicolas Kylilis, what a douche!
-
*Gel electrophoresis to determine concentrations of purified fragments
+
*Diluted my overnights 250μl into 5ml to grow up to around 0.5 OD
-
*Dephosphorylation of pSB1C3
+
*Gonna perform assays with them this afternoon after some growing time :)))))))
-
*Ligation of pSB1C3 with lytC overnight
+
*Doing data analysis with my last assay results comparing high copy (1C3) vs low copy (3K3)and the effects on XylE activity..boring
-
*Transform ''E. coli'' with pVeg ligations (has to be repeated)
+
*Finalising the polo-shirt logo design, it's looking fiiiine, we're gonna look goooood ;)
-
||
+
'''Tues 12th Oct'''
 +
*Transformations worked! we now have pVeg-XylE in 3k3 and reverse-XylE with lacI promoter (C-TEV vector)i did replica plates, catechol assay plates and overnights for minis. Will mini prep tmro and transform them into TOP10 and we should be good to go with all constructs.
 +
* Assays messed up, my fault :( 3k3 didnt respond to catechol, so i think that I picked a background colony, i wont pick it again. The results are a bit ridiculous and will probably be chucked.
-
*Transform E. ''coli'' with the ligated pSB1C3-lytC (from K5 culture)
+
'''Weds 13th Oct'''
-
*Transform E. ''coli'' with the ligated pSB1C3-pVeg
+
mini prepped 3k3-pVeg-XylE-B0014 and went to the school workshop! set off overnight midi of culture 9
-
*Repeat PCR amplification of pVeg from pSB1AK3 with the shorter extension time
+
-
||
+
'''Thurs 14th Oct'''
-
 
+
Midi prepped 3k3-pVeg
-
*Gel electrophoresis to analyse PCR
+
-
 
+
-
||
+
-
 
+
-
*Restriction digest of pSB1C3-lytC with
+
-
# XbaI and SpeI
+
-
# AccI
+
-
*Gel electrophoresis of digest
+
-
 
+
-
||
+
-
 
+
-
*Restriction digest of pSB1C3-pVeg, pSB1C3-lytC and linker sequences in pSB1C3 with EcoRI and SpeI.
+
-
*Gel electrophoresis of digests
+
 +
'''Fri 15th'''
 +
Transformed all my constructs into T0P10 3k3-J23101/pveg and PSB1C3-J23101/pVeg
|}
|}
-
 
+
{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20"
-
====Monday, 13th-Sep-2010====
+
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"| Output Photo Gallery
-
 
+
-
* The 3 minipreps of K5 were made.
+
-
* Two mini preps were analysed using a restriction digest with XbaI and SpeI.
+
-
* pSB1C3-B0014 was also digested with XbaI and SpeI to remove B0014.
+
-
* Gel electrophoresis was used to confirm correct restriction and the bands were then gel purified.
+
-
* After gel purification the relative concentration of vector to insert were determined by gel electrophoresis.
+
-
* After dephosphorylation of pSB1C3, overnight ligation was set up.
+
-
* ''E. coli'' were transformed with pSB1C3-pVEG but could not be plated so transformation will have to repeated.
+
-
 
+
-
====Tuesday, 14th-Sep-2010====
+
-
 
+
-
*The meeting with Prof Fenwick and Dr Harrison from the Schistosoma Control Initiative (SCI) was very rewarding. Feedback can be found on the Human Practices page.
+
-
*We performed two transformations of E. ''coli'' with:
+
-
#the ligated pSB1C3-lytC (from K5 culture)
+
-
#the ligated pSB1C3-pVeg
+
-
*We also repeated the PCR amplification of pVeg from pSB1AK3 with the shorter extension time, which produced a much higher concentration of pVeg so it will be easier to ligate it with pSB1C3.
+
-
 
+
-
====Wednesday, 15th-Sep-2010====
+
-
 
+
-
*Transformations of E. ''coli'' with pSB1C3-pVEG and pSB1C3-lytC was successful.
+
-
*Mini prep cultures were set up for both pSB1C3-lytC (7) and pSB1C3-pVeg (5).
+
-
*The team had a meeting to discuss the wiki.
+
-
 
+
-
====Thursday, 16th-Sep-2010====
+
-
 
+
-
*The mini preps of pSB1C3-lytC were successful, however the mini preps of pSB1C3-pVeg failed.
+
-
*Restriction digest of pSB1C3-lytC with XbaI and SpeI for the double digest, as well as AccI for the single digest were performed.
+
-
*Cultures for new pSB1C3-pVeg mini preps were set up.
+
-
*Unfortunately gel electrophoresis of the digestion of pSb1C3-lytC were not conclusive because Either XbaI or SpeI did not cut, nor did AccI. Therefore the digest will be repeated tomorrow with EcoRI rather than XbaI.
+
-
 
+
-
====Friday, 17th-Sep-2010====
+
-
 
+
-
*Mini preps of pVeg were successfully made
+
-
*Restriction digest of pSB1C3-pVeg, pSB1C3-lytC and several linker sequences also in pSB1C3 were set up using EcoRI and SpeI.
+
-
*Gel electrophoresis was used to analyse the restriction digest. It showed that pSB1C3-pVeg as well as the linkers had been ligated and transformed into E. ''coli'' successfully. However we most likely did not succeed in construction pSB1C3-lytC yet.
+
-
 
+
-
 
+
-
 
+
-
===Week 12===
+
-
 
+
-
{| class="wikitable" style="text-align: center; width: 80%; height: 170px;" border="1"
+
-
 
+
|-
|-
-
  ! Week 12 !! Monday, 20th-Sep-2010 !! Tuesday, 21st-Sep-2010!! Wednesday, 22nd-Sep-2010 !! Thursday, 23rd-Sep-2010 !! Friday, 24th-Sep-2010
+
|[[Image:IC_IMAG0310.jpg|thumb|center|300px|First Successful catechol assay :)))]]
 +
|[[Image:IC_IMAG0313.jpg|thumb|center|300px|First successful catechol assay]]
|-
|-
-
  | '''MORNING''' ||
+
|[[Image:IC_CS1.JPG|thumb|center|300px|Experiment which determined the yellow product to be exported out of the cells into the media and not retained by the cells]]
-
 
+
|[[Image:IC_CS2.JPG|thumb|center|300px|Cell vs supernatant assay]]
-
*Prepare K5M3 (lytC in blunt ended ligation vector) as well as H2 and H3 for sequencing
+
|-
-
*Analysed digestion of H2 and H3 with AseI
+
|[[Image: IC_IMAG0322.jpg|thumb|center|300px|Cuvette assay set up]]
-
 
+
|[[Image:IC_IMAG0315.jpg|thumb|center|300px|:-)]]
-
||
+
-
 
+
-
*Mini prep of Gly-X Com CDE 1-2
+
-
 
+
-
||
+
-
 
+
-
*Midi prep cultures set up yesterday were used to make successful midi preps of pSB1C3-pVeg
+
-
 
+
-
 
+
-
||  
+
-
 
+
-
-
+
-
 
+
-
||  
+
-
 
+
-
*Mini prep of ComD from the cultures set up yesterday
+
-
*Start midi prep of the linkers from the cultures set up yesterday
+
-
 
+
|-
|-
-
  | '''AFTERNOON''' ||
+
|[[Image:miaow.jpg|thumb|center|300px|]]
-
 
+
|[[Image:miaow2.jpg|thumb|center|300px|]]
-
*Meeting with supervisors for track selection and abstract
+
|-
-
*Mini prep cultures and replica plate set up for Gly-XCom AB
+
|[[Image:miaow3.jpg|thumb|center|300px|final batch of transformed XylE constructs into TOP10s]]
-
 
+
|[[Image:miaow4.jpg|thumb|center|300px|Fusion GFP-XylE vs XylE cuvette response assay]]
-
||
+
|-
-
 
+
|[[Image:miaow5.jpg|thumb|center|300px|View from above]]
-
*Restriction digest of the linkers with EcoRI and SpeI as well as AccI
+
|[[Image:miaow6.jpg|thumb|center|300px|Plate after assay!]]
-
*Gel electrophoresis
+
-
*Set up midi prep cultures for pSB1C3-pVeg (H2,H3)
+
-
 
+
-
||  
+
-
 
+
-
*Restriction digest of 3 mini preps of Flex Com CDE using EcoRI and SpeI for double and AccI for single digest.
+
-
*Gel electrophoresis of Flex Com CDE restriction fragements
+
-
 
+
-
||
+
-
 
+
-
*Determine concentration of DNA in pSB1C3-pVeg midi prep
+
-
*Set up midi prep cultures of the linkers that were sucessfully tested
+
-
*Make replica plate and set up mini prep cultures of pSB1C3-ComD
+
-
 
+
-
||
+
-
 
+
-
*Restriction digest of ComD mini preps with EcoRI and SpeI
+
-
*Finish midi preps of linkers
+
-
*Measure midi prep DNA concentrations
+
|}
|}
-
 
-
===Monday, 20th-Sep-2010===
 
-
 
-
*Samples of lytC in the blunt-ended vector (K5M3) as well as pVeg (H2, H3) were send off for sequencing. No primer had to be added to K5M3 because it is avaliable at the company.
 
-
*We analysed the restriction digest from yesterday using gel electrophoresis. AseI did not cut either sample of pVEG which is a positive result because AseI does not cut pEVG but BOO14, allowing us to say with some confidence that we have got pVEG. Sequencing will give us certainty soon.
 
-
*Replica plate and mini prep cultures for the linker Gly-X Com AB were set up
 
-
 
-
===Tuesday, 21st-Sep-2010===
 
-
 
-
*The cultures of Gly-X Com CDE 1-2 were used to prepare mini preps.
 
-
*The previously made mini preps of linkers as well as today's were analysed with a restriction digest using EcoRI and SpeI for a double and AccI for a single digest.
 
-
*Gel electrophoresis of the restriction fragments showed that many, but not all of the samples, appear to have worked well. HEL-TEV 1-3 have worked, Gly-X Com CDE 2-3 have worked, Flex Com CDE 1-3 all failed so far, but more mini prep samples will be tested tomorrow, Flex TEV 2 worked, and both Gly-X Com AB 1-2 were successful too.
 
-
*Midi prep culture for pSB1C3-pVeg (H2,H3) were set up.
 
-
 
-
===Wednesday, 21st-Sep-2010===
 
-
 
-
*Overnight cultures (H2,H3) of pSB1c3-pVeg were used to make midi preps.
 
-
*Another 3 mini preps of the linker Flex Com CDE were anaysed with a restriction digest using EcoRI and SpeI for the double and AccI for the single digest.
 
-
*Gel electrophoresis of the restriction fragements showed that the ligation/transformation has not been successful, so it will be repeated.
 
-
*Sequencing results confirmed, that pSB1C3-pVeg has the correct sequence. Midi preps concentration will be measured tomorrow.
 
-
 
-
===Thursday, 22nd-Sep-2010===
 
-
 
-
*Determine concentration of DNA in midi prep of pSB1C3-pVEG:
 
-
#Concentration of DNA in H2 was ~950ng/µl
 
-
#Concentration of DNA in H3 was ~430ng/µl
 
-
*We set up midi prep cultures of the linkers that have been successfully mini preped and tested by a diagnositc digest.
 
-
*We set up mini prep cultures as well as a replica plate of the pSB1C3-ComD.
 
-
 
-
===Friday, 23rd-Sep-2010===
 
-
 
-
*A mini prep of pSB1C3-ComD was made successfully.
 
-
*Subsequent analysis by restriction digest with EcoRI and SpeI confirmed that ComD is in the vector in the correct orientation.
 
-
*The midi preps of the linkers Hel-TEV, Flex TEV, Gly-X ComCDE and Gly-X Com AB were made and the concentration determined.
 

Latest revision as of 03:25, 28 October 2010

Lab Diaries Overview | Surface Protein Team | XylE Team | Vectors Team | Modelling Team
Here are the technical diaries for our project. We've split them up into three lab teams and the modelling team. We think it's really important that absolutely anyone can find out what we've been doing. For a really detailed look at what we did, and when, you've come to the right place!
XylE Team

Objectives:

  • construction of XylE fusion protein
  • Testing expression of XylE in E.coli and characterization under the control of a constitutive promoter
  • Construction of the -ComE promoter/XylE fussion protein- expression system
  • Construction of the -LacI promoter/XylE fusion construct- expression system


Here's a picture of the final construct:

IC Module3.JPG
Typical XylE assay
Week 6

Week 6 Monday Tuesday Wednesday Thursday Friday
Morning
  • mini-prep kit of XylE-transformed ''E.coli'' (already overnight grown)
Afternoon
  • Starting of “Testing expression of XylE in ''E.coli''” objective
  • 1)Annealing EcoRI and speI oligos to J23101 promoter which will be annealed later in front of the RBS-XylE registry gene (overnight)
  • gel analysis of mini-prep derived XylE plasmid.(requires first digestion of the vector with restriction enzymes)


Thursday, 12-Aug-2010

  • Annealing DNA strands of J23101 promoter in a water bath

we constructed the standard E.coli promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assembly to construct a promoter-RBS-XylE design in a psB1C3 vector. E.coli will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted BioBricks.

  • Prepared two overnight cultures of XylE transformed E.coli (one 50microliters and one of 450μl)

these cultures are going to be used tomorrow for mini prepping. Mini prep will allow us to isolate E.coli 's plasmid DNA(which contains the XylE gene).

Friday, 13-Aug-2010

  • Mini-prep of XylE transformed E.coli

Mini prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasmid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini prep will later be used for the midi-prep (that gives out higher yields of DNA needed for cloning).

  • Gel analysis of plasmid DNA retrieved from mini prep of XylE transformed E.coli, cut with restriction enzymes. From light to the left, 50μg digested DNA : 50 undigested DNA : 450 digested DNA : 450 undigested DNA. In lanes 1 and 3 the smaller band has a size of about 1kB which corresponds to RBS-XylE gene. The bigger bands are the cut vectors. In lanes 2 and 4 is the uncut BioBrick from the registry. It appears smaller on the gel than it actually is as circular DNA travels faster through the pores of agarose gel rather than linearised DNA.
Week 7
IC 10-08-13 Digest image.jpg

Week 7 Monday Tuesday Wednesday Thursday Friday
Morning
  • midi prep XylE ''E.coli'' (2hrs)
  • gel extraction kit on XylE gene trapped in agarose
  • 3A assemply: make replica plates (overnight)
  • Catechol assay of ''E.coli''
  • Mini prep XylE ''E.coli''
  • Midi prep XylE ''E.coli''
Afternoon
  • restriction digestion of XylE for 3A assemply
  • gel purification of XylE from restriction digestion
  • 3A assembly of vector, XylE and J23101 promoter
  • above construct transformed in ''E.coli''
  • primers arrived!
  • PCR extension of XylE and GFP, round 1


Monday, 16-Aug-2010

  • Midi prepped the XylE-transformed E.coli. The DNA yield from the midi prep was 134μg as determined by spectrophotometry. This is the XylE that is going to be used for all further experiments.
  • Restriction digestion of midi prepped XylE by Xbal and PstI to prepare it for 3A assembly. (with J23101 promoter and PSB1C3 vector)
  • Gel analysis of the restriction digestion mixture to isolate XylE gene


Midi prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).

  • I made an overnight culture of Bacillus
Midi prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).

Tuesday, 17-Aug-2010

  • Using the Gel Extraction Kit, we isolated the restriction enzyme cut XylE gene from the agarose gel band.
  • Gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine their ratios for 3A assembly ligation
  • 3A assemply of XylE, J23101 promoter and pSB1C3 vector.
  • Transformation of XL-Blue competent E.coli with the above construct.
gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation

gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation

  • I followed Chris's Bacillus transformation protocol to transform Bacillus with constitutive GFP and RFP DNA as well as a control without DNA
Successful Bacillus transformation!


Thursday, 19-Aug-2010

  • We run the XylE-GFP1 PCR reaction to construct the His-GFP-Flag and Linker-XylE-Spe construct.

Friday, 20-Aug-2010

The J23101 gene in a BioBrick vector containg RFP gene

  • We run a gel on XylE-GFP1 PCR reaction. Results: GFP was extended successfully, XylE extension FAILED (too much non-specific annealing)
  • Catechol assay on 2hrs bench ligation of promoter, XylE and vector failed.
  • Two replica plates of overnight ligated J23101, XylE and pSB1C3 transformed E.coli (one for catechol assay)
  • The His-GFP-Flag DNA was gel purified
  • Transformation of XL-1Blue cells with J23101 in J62001 vector from the registry. One more attemp to construct a successful promoter-XylE ligation, since we believe that the strand annealed promoter was of bad quality
Week 8

Week 8 Monday Tuesday Wednesday Thursday Friday
Morning
  • PCR extention of His-GFP-flag round 2
  • midi prep registry obtained J23101
  • gel purification of XbaI-His-GFP-flag-TEVs
  • gel analysis of overnight ligation and gel separation
  • gel analysis of linker-XylE-SpeI PCR reaction
  • make replica plate and catechol assay plate
  • catechol assay
Afternoon
  • Gel purification of linker-XylE-Spe PCR construct
  • Gel analysis of His-GFP-flag round 2 PCR rection and then gel separation of DNA
  • restriction digestion, gel analysis and gel purification of registry obtained J23101
  • PCR extension of linker-XylE-SpeI
  • ligation of pSB1C3, J23101 and XylE (overnight)
  • gel purification of overnight ligation
  • transformation of ''E.coli'' with overnight ligation product and selection by plating
  • gel purification of TEVs-linker-XylE-SpeI construct
  • PCR construction of fusion protein
  • Gel analysis and gel purification of fusion XylE protein

Monday, 23-Aug

  • Set up overnight cultures for midi prep
  • Gel separation of linker-XylE-Spe DNA FAILED
  • PCR reaction for extension of His-GFP-Flag
  • Catechol assay on E.coli transformed with overnight ligated J23101, XylE and pSB1C3. FAILED

Tuesday, 24th-Aug

  • Midi prepped registry obtained J23101. A yield of 130ng/μl of promoter was obtained. The promoter is in a BioBrick vector called J62001. The promoter is upstream of RFP gene.
  • the vector carrying the promoter was digested with SpeI and PstI, while XylE gene was digested with XbaI and PstI.
  • The promoter and the XylE gene were gel purified.
  • A reaction between the promoter(still on vector) and XylE was set on for overnight ligation
  • PCR purification of GFP2 -> GFP construct ready for full fusion protein.
  • Gel purification of XylE lost DNA along the way. Thus PCR XylE1 with gradient for temperature scanning (taq): PCR round 1 included 62°C and only rev primer to create pool of successful extensions with with rev primer (60-62°C). PCR round 2 with Fwd primer and temperature scale (68-68°C, 72-74°C).

Wednesday, 25th-Aug

Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appalling (explanation for Nick: really bad) results, 2nd gel run was successful.

  • Performed a ligation reaction between the vector containing J23101, and XylE (one on bench and one overnight one).
  • Transformation of the new plasmid into competent E.coli. Successfully transformed colonies can be selected for by loss of RFP expression.
  • XylE-1 PCR with temperature cascade. Gel analysis and purification.

Thursday, 26th-Aug

  • White colonies from the promoter-XylE transformed E.coli were picked and transferred to new amp plates. One is the replica plate and the other is the catechol assay plate.
  • XylE-1, two rounds of PCR/purification were run to obtain a sufficiently clear band. An additional PCR run for XylE-1 was discarded afterwards.

Friday, 27th-Aug

  • Catechol assay performed on promoter-XylE transformed E.coli. SUCCESSFULL
Plate before adding catechol assay
After addition of catechol colonies turn yellow-orange in seconds!!
  • XylE-2 PCR and gel-purification cycles (2x) to obtain clear band. XylE-2 is now ready for assembly of the GFP-XylE fusion protein.

Saturday, 28th-Aug

  • Preparing the annealing step between the GFP-2 and XylE-2 constructs, we discovered sequence dissimilarities in the TEV-cleavable regions which we planned to use for the annealing step. Nevertheless a PCR was run with appropriate conditions (allowing for a minimal amount of unspecific annealing).

Sunday, 29th-Aug

  • Gel analysis of the attempted annealing reaction of GFP-2 XylE-2 showed unsufficiently clear bands for gel-purification. A new reaction is being prepared: 10 rounds of annealing PCR, followed by addition of primers (5' primer for GFP-2 and 3' primer for XylE-2) in order to introduce an amplification step in the reaction. --- Following Kirstin's advice, we are discarding this reaction and wait for the arrival of new primers for XylE-2 (5' + TEV) and GFP-2 (3' +TEV).
Week 9

Week 9 Monday Tuesday Wednesday Thursday Friday
Morning
  • mini prep of promoter-XylE transformed ''E.coli''
  • design of reverse primer for GFP to add the corrected TEV sequence to the construct.
  • midi prep of promoter-XylE transformed ''E.coli''
  • restriction digestion of pSB1C3+terminator vector DNA
  • restriction digestion of promoter-XylE DNA
  • Gel purification kit of cut promoter-XylE DNA
  • Preparation of M9 and LB medium for assay
  • Gel analysis of of insert and vector DNA
  • find optimun wavelength for catechol assays
  • find optimun cell density and catechol concentration for assay
  • Reverse primer for GFP with modified TEV arrived - primer dilution
Afternoon
  • gel analysis of mini prepped promoter-XylE
  • cross-check for primer design (GFP-TEV-2) and ordering
  • PCR purification of cut pSB1C3+terminator vector DNA
  • Gel analysis of cut promoter-XylE DNA
  • dephosphorylation of the vector+terminator
  • ligation reaction between promoter-XylE insert and pSB1C3+terminator vector
  • GFP 2 PCR with new reverse primer (adopted TEV sequence)


Monday, 30th-Aug

  • Mini prep of 4 x J23101-XylE taken from 4 different colonies (8 14 24 27).
  • The gel analysis showed successful vector uptake.
  • Set up an overnight culture for midi using colony 24 (gel analysis showed similar results colony 24 was picked randomly.)
  • A new primer was designed in order to add a corrected TEV-protease-cleavable sequence to the His-GFP-Flag construct. This was controlled and ordered.

Tuesday, 31st-Aug

  • Midi prep of colony 24 for XylE-J23101 the final concentration was ~100ng/μl which wasnt so great but Chris says the protocol produces very poor yields.
  • We are performing the next building step of our vector. PSB1C3 containing terminator B0014 was cut with EcoRI and XbaI. The insert was cut with EcoRI and SpeI and both were incubated for 1.5hrs. Wolf is now running a gel to purify out the insert via gel purification and perform a PCR purification on the vector.
  • Advisors have decided it's best not to use Jeremy's tagged XylE due to the 93% difference. Kirsten will be tagging the registry XylE and we shall purify and assay with that instead.
  • We shall see purification expert Kieran tomorrow and talk through the process. Chris will also talk us through our characterization of XylE experiments- we will use the robot after it's been programmed but until then we can use the plate-reader.

Thursday, 2nd-Sept

Spectra of XylE transformed E.coli after addition of catechol assay. The broad peak around 380nm wavelength arises is due to the presence of the product of the enzymatic reaction involving pyrocatechol and XylE enzyme. This peak if absent if a culture of XylE transformed cells are measured without the addition of catechol
  • Spectrophotometry experiments with XylE transformed E.coli in LB medium (M9 culture was contaminated) reveiled the followings: On catechol assay of the trasformed cells, the positive yellow output can be quantitively measured by a broad peak at 380nm.
  • Transformation of competent E.coli cells with promoter-XylE-terminator pSB1C3vector.

Friday, 3rd-Sept

  • experiment to determine concentrations of catechol and cell density for assays
  • The new GFP + TEV primer arrived, was diluted and used to set up the appropriate PCR.


Catechol assay on XylE-trasformed cells in a 96-well plate (A to H decreasing cell concentration, 1-10 decreasing catechol concentration, column 11 and 12 negative and control)
Week 10

Week 10 Monday Tuesday Wednesday Thursday Friday
Morning
  • PCR extension of PVeg promoter
  • Gel purification of extended PVeg promoter
  • Annealing PCR for GFP-XylE fusion protein
  • vector with insert tranformed into ''E.coli'' and plate to select for successful transformation
  • prepare overnight cultures of pVeg-RBS transformed ''E.coli''
  • Gel-analysis of amplification-PCR for XylE-2
  • midi prep of PVeg-RBS vector
Afternoon
  • Gel analysis and purification of the GFP-TEV construct
  • restriction digestion and gel purification of thr extended pVeg promoter
  • overnight ligation of the cut pVeg promoter in a vector
  • Gel analysis of GFP-XylE annealing PCR - unsuccessful reaction
  • Amplification PCR for XylE-2
  • Gel-purification of amplification-PCR for XylE-2


Monday, 6th-Sept

  • PCR extension of pVeg promoter: EcoRI---pVeg---RBS-SpeI
  • I performed a catechol assay on the picked transformed colonies to deduce which ones were successfully transformed with the insert plus vector. 1-5 7 and 10 failed to turn yellow (1-5 were background controls)leaving 8 yellow colonies.
  • I had to perform a colony PCR on two selected colonies 8 and 14 to check the correct insert+terminator was present.
  • Colony 8 when run on a gel analysis showed the correct size 924 XylE + 97 J23101 + 35 B0014.
  • This was set up as an overnight culture.
  • Gel analysis of the GFP-TEV construct showed satisfactory bands.

Tuesday 7th Sept

  • Mini prep of the overnight Colony 8 culture (by Wolf)
  • Midi prep of overnight culture
  • Gel purification of PCR product EcoRI--pVeg--RBS--SpeI
  • Overnight restriction digestion of EcoRI--pVeg--RBS--SpeI with SpeI
  • Started data analysis of plate assay
  • Annealing PCR reaction included 2 samples without and one with additional primers (XbaI-His-GFP Fwd, SpeI-XylE Rev). While the primer-including reaction did not show any clearly identifiable bands, the others showed clear, if very weak bands at 800 and 1000bp, which represent the GFP and XylE constructs respectively. No band was identifiable at the 1.7 kb range, which would have indicated a successful annealing reaction. However, problems with the lense of the gel-analyser were only discovered later to have severely reduced the band brightness. Potentially a PCR-reaction with appropriate primers to amplify an annealed product(XbaI-His-GFP Fwd, SpeI-XylE Rev), could have been successful. However the reaction mixture was disposed of before this became clear.

Wednesday, 8th Sep

  • A second PCR extension of pVeg promoter to introduce the RBS and cut sites. It was also gel purified and stored in gel lumbs in the freezer. (maybe needed later so keep in mind).
  • Overnight restriction digestion completed. Then, it was run on the gel to check if it worked and then gel purified again.
  • Vector PSBI-C3 was digested to remove the terminator and make it sticky for the insert (pVeg-RBS). Then it was run on the gel to check if restriction has worked, but the gel didn't run far enough to determine easily between undigested and digested vector.
  • For further annealing reactions for GFP-XylE constructs additional XylE(2) template was required -> amplification PCR for XylE(2).

Thursday, 9th Sep

  • Gel with digested and undigested vector PSBI-C3 was run and then the digested vector was purified.
  • Gel was run to determine the DNA concentration ratio for the ligation of PSBI-C3 and pVeg-RBS.
  • Vector PSBI-C3 was dephosphorylated.
  • Ligation of PSBI-C3 and pVeg-RBS has been set up overnight.
  • Gel-analysis and gel-purification of the XylE(2) amplification PCR product.
  • The transformation of E.Coli with pVeg-RBS in PSBI-C3 and PSBI-C3 by itself (to check see how successful dephosphoryaltion of PSBI-C3 was and estimate the percentage of bacteria that contain the insert) was completed
  • Concentration of the midi prep of J23101-XylE-B0014 was determined to be ~600ng/μl (using new protocol)
  • Kyasha kindly digested my midi with EcoRI and SpeI and performed a gel analysis. The results show a potential additional plasmid contaminating my midi however the concentration of DNA was extremely high. NB Chris said that it could be sheared DNA from a midi prep step.
  • The midi prepped plasmid was transformed into testing E.coli strain TOP10.

Friday, 10 sep

The transformation was a SUCCESS. 2x replica plates were made plate 1# 1-6 plate #2 6-11; colony 6 and colony 9 of plates 1 and 2 respectively were transfered into a 5ml liquid culture + 5μl CmR. These will later be turned into glycerol stocks. After the replica plates have grown up mini preps on a number of colonies shall be performed - this hopefully will eliminate the contaminating plasmid DNA. This will be followed by a midi prep.

Saturday, 11 Sep

The transformation of E.Coli with PSB1-C3 with insert did not work :(

Week 11

Week 11 Monday Tuesday Wednesday Thursday Friday
Morning
  • Gel-analysis of amplification-PCR for XylE-2
  • midi prep of pVeg-RBS vector
  • In preparation of ''catechol cell-death assay'' tranformation of Top10 cells with CMR plasmid (psb1C3)
Afternoon
  • Gel-purification of amplification-PCR for XylE-2
  • plating and overnight incubation of transformed top10 CMR
  • overnight cultures for catechol cell death assay: Top10 cells XylE/CMR

Monday 13th september Overnights weren't set up on sunday so they were made up alongside some assay cultures. J23101-XylE-B0014 colonies 8 and 10 of the replica plate #2 were picked. Chris also provided a replica plate containing 3k3 vector colonies. This was over a year old and he was unsure whether it was the correct plasmid or if the cells would grow up. I picked all available colonies 102 150 151 and 260 of kanamycin resistance. Lastly for assays 2x LB 2xM9 cultures were made 5ml +5ul antibiotic.

Tues 14th September

test confirming that yellow product of XylE enzmymatic reaction is leaks back from the cytosol into the solution.1C and 2C is the XylE producing cells after centrifuging and redissolving of the pellet and 1S and 2S is the supernatant after centrifuging
  • assay on plate reader of:
    • 0-0.75 initial O.D. of transformed E.coli
    • 0-1mM initial catechol concentration

The assay was carried out with E.coli, top ten spcies, transformed with J23101-XylE-B0014 in pSB1C3 vector.The overnight culture wastransfered in new medium this morning for 4 hrs before assaying. LB medium was used for dilutions and blank. Catechol was diluted in ddH2O. Data analysis of the assay

  • Mini preps of XylE 8,10 colonies and 3K3;102 151 260 colonies. Analytical digests were performed using E+S on XylE, E+S AccI and HindIII+E for 3K3 vector respectively. The band patterns correctly identified the vector to be 3k3. Colony 10 of XylE and Colony 260 of the 3k3 vector were set up direct into 100ml LB and so will require til 12pm for midi prepping on weds. Nick performed a second plate reader assay to determine the minimal concentration of Catechol to use for assays. I took 990ul M9 culture containing colony 10 and 10ul catechol was added. This was incubated for 10 mins and then spun down. The supernatant was removed into a cuvette and the cells resuspended in M9 salts. The ODs were then read on the spectrophotometer at 380 and 600nm. It was found that control cuvette of M9 salts had a miniscule reading. M9 + cells ~0.007. Supernatant + cells were ~ 2.5 therefore we could deduce that the coloured catechol breakdown product is exported out of the cells.

Piotr:

  • Mini prep of 1 sample (6 samples lost due to mistake) of GFP-Xyle fusion protein and its digestion with Spe and Xba (gel to be run on the next day)
  • Preparing E.Coli colonies for the next day for mini prep to be redone
  • PCR of 6 samples of GFP-Xyle fusion

Weds 15th September

  • Midi preps of overnights.
  • In preparation for assays determining the effect of catechol and/or breakdown product on cell viability we prepared Top10 cells, one strain containing pVeg-XylE-terminator and the other containing a CMR plasmid of similar size. As the XylE cells have already been prepared, only Top10 transformation with CMR-plasmid had to be carried out.

Thurs 16th September

  • Top10 CMR transformation was successful. Four overnight cultures of each Top10 XylE and Top10 CMR were set off with either M9 or LB medium respectively.
  • Piotr: Did mini preps from bacteria with blunt ended Xyle-GFP fusion and did diagnostic using both:

- digest with XbaI and SpeI

- PCR reactions with the following primers added: HIS-GFP, XylE-Xba and the other sample with HIS-GFP and GFP-flag

The PCR reactions were not very conclusive on the gels but digests allowed to determine that colonies 1->5 seem to have the right sizes of DNA in them and on Friday they will be prepared to be sent off for sequencing

  • Continuation of the midi from overnight step. A gel analysis was run which showed the correct bands were present. A gel purification of the protein was then performed and the bands excised.

Friday 17th September

  • Catechol assay of transformed E.coli Top ten(J23101-XylE-terminator) in M9 medium + data analysis

The gel purifications of 3k3 vector and XylE were used for ligation. 3K3 was dephosphorylated and then ligated in a ratio 5:1 with the insert. This was left overnight and Chris tried to transform with it on sunday.. ligation and transformation failed :-(

Week 12

Monday 20th Sept Performed ligation again. Errors in the sequencing we received back regarding J23101-XylE-B0014. One was a log error (in XylE) phew. The other is in PSB1C3 out of the scar site and should be OK. Chris is currently assembling the pVeg promoter+ RBS in a vector. Once he has finished this, I will combine XylE-GFP and XylE with this promoter for comparison characterization with J23101 (in E.coli and Bacillus)

Tues 21st Sept

  • Ligation to be transformed in a bit. (Issue with the vector not being cut efficiently=failed ligations?)
  • I am cutting the original XylE midi as well as PSB1C3 containing B0014 and ligating them today (hopefully) the vector is purified, waiting on the gel run and then extraction of XylE insert. A ligation ratio must be determined from a gel analysis then dephosphorylation of the vector and then ligation. This will produce a promoter-less XylE+terminator vector which will be readily available to switch in the desired promoter (E+S cut)
  • Florian is performing a mini prep on pVeg promoter which if it is correctly identified in the gel i can use to ligate to my XylE + PSB1C3-B0014.
  • Piotr is PCRing the reverse version of XylE that will be under the control of inducible promoter LacI (not delivered from synthesis yet) that will become a testing construct.

Wed 22nd Sept

  • Piotr's PCR has been successful for 3 samples (1. recommended annealing temp; 2. recommended + 2 degrees and 3. recommended + 4 degrees). Sample no1. has been run on the gel and gel purified. It has been cut overnight with SpeI and tommorrow in the morning XbaI will be added.
  • I (Maddie!) performed steps towards the ligation (XylE-3K3) again from scratch (inefficient cutting steps??) including digest, gel analysis and extraction.
  • I ligated PSBC3-B0014 and XylE
  • I picked successfully transformed colonies for a replica plate and set up overnight mini cultures of 1,2,3 ComC DE promoter FWD and 4,5,6 ComC promoter Rev

Thurs 23rd Sept First day of AUTUMN!!

  • I determined the ligation ratio for XylE and 3k3 (1:3) performed dephosphorylation of vector then ligation, to be transformed along with the other ligation tomorrow hopefully.
  • I did mini preps of the Comc DE promoters, performed a ES test digest and am currently waiting for gel results. I shall then set up overnight midis.
  • Miniprep results were fabulous, Ive chosen colony 1 and colony 4 which are ComCDE promoters FWD and REV respectively. They are made up into overnight midi cultures for tomorrow.

Fri 24th Sept

  • Midi preps of ComC DE promoters FWD and REV (colonies 1,4)
  • to be followed by a digest. XbaI Pst for FWD (not known for the reverse currently) to insert XylE after it.
  • Ligation with XylE and PSB1C3-B0014 possibly.
  • Transformation of XylE-PSB1C3-B0014 and of XylEe-3k3 into E.coli later
  • Hi Nick, hope revision is going well. Love from Team XylE xox
Week 13

Monday 27th Sept

  • Successfull transformations: now have XylE-PSB1C3-B0014 (no promoter) and 3K3-J23101-XylE-B0014. No background plate colonies..many colonies for 3K3-XylE and a few for PSB1C3-XylE. Colonies have been replica plated (plus catechol assay plate for 3K3-XylE) and set up for mini prepping tomorrow.
  • Sumo tagged XylE will be given to us by Kirsten at some point this week and we will have to purify it. This can then be used for in vitro testing and comparisons made with the linker-XylE protein.
  • pVeg and ComC DE promoter FWD can be added into the promoter-less construct after it has been midi prepped and digested accordingly. ComC DE promoter REV can be added onto the reverse XylE construct (PCR'd by Piotr) AFTER it has been put into PSB1C3 and is more manageable. Kirsten is taking care of the GFP-XylE fusion for now, including putting the pVeg promoter plus RBS infront of it (proving difficult.) We still need to receive LacI promoter to create the inducible test version of the fusion XylE-GFP protein.

Tues 28th Sept

  • Performed mini preps and then E+S / E+S AND AseI digests of colonies 1-7 3k3-XylE and 1-3 C3-B0014-XylE respectively. Gel runs showed them to be the correct thing :)))))) so, 3k3-XylE final vector is now left as 2-7 minis (catechol assay test showed colony 1 was background so DO NOT USE) in my DNA box (orange tubes). Colony 1 of the C3-B0014-XylE was selected for midi prepping and will be performed tomorrow.

Weds 29th Sept

  • Midi prep of XylE-GFP fusion protein along with C3-B0014-XylE. The C3-B0014-XylE midi will then be used in 2x cloning digests (E+S) to insert pVeg and ComC DE FWD promoters. These will be obtained and also digested E+S to get out of their current vectors a gel analysis and purification will be run of all 3 in parallel, followed by a ligation ratio gel analysis, dephosphorylation of vector then ligation.
  • Primers for J23101-XylE arrive today so they can be used to PCR out J23101-XylE (From my J23101-Xyl-E midi) with blunt ends and this will be ligated into the final Spec vector so that we can get some data with Bacillus.
  • J23101-XylE-B0014 XylE-3K3 and PSB1C3-B0014-XylE were all sent off for sequencing

Thurs 30th Sept

  • I gel purified ComCDE FWD promoter, C3-XylE-B0014, pVeg vector and XylE-B0014. Did a gel analysis to get ligation ratio.
  • Set up midi culture of J23101-XylE-B0014 colony 8
  • Set up midi culture to repeat GFP-XylE midi prep
  • Set up 6x minis (plus replica plate) of final Spec colonies 1-6
  • PCR reaction using newly arrived primers to get blunt ended J23101-XylE.

Friday 1st Oct

  • morning maddie, from earl's court! :)

ahahahaha awesome. hey you :)

  • Today I peformed ligations for ComCDE FWD promoter and PSB1C3-XylE-B0014 and also PSB1C3-pVeg and XylE-B0014 they will be left overnight and transformed by chris tmro thaaanks.
  • I ran the PCR Kirill performed yday to get out blunt ended J23101-XylE on a gel; there was no template DNA! so I've just set up the reaction again... hopefully i'll purify it later for ligation into the final Spec vector that kyasha has maaaaade :))) then we can test XylE in Bacillus.
  • I'm currently performing Midi's of my J23101-XylE-B0014 (running low) and of GFP-XylE (3rd time!)

have a look :) it's from John Hoppkins wiki 2008. There are one or two good ones!

Love: Before I heard the doctors tell The dangers of a kiss;

I had considered kissing you. The nearest thing to bliss.

But now I know biology and sit and sigh and moan;

six million mad bacteria and I thought we were alone!


Biology is the science of moving tiny droplets of invisible liquids from one tube to another.


The human body was designed by a civil engineer. Who else would run a toxic waste pipeline through a recreational area?


I have a hunch that the unknown sequences of DNA will decode into copyright notices and patent protections.


It has recently been discovered that research causes cancer in rats.


A mouse is an animal that, if killed in sufficiently many and creative ways, will generate a PhD.

Week 14

Monday 4th Oct Did a replica plate and catechol assay plate/ minis 2 6 7 9 12 16. (Successful pVeg-XylE transformation)

Tues 5th Oct 4 16 colonies were background the rest turned yellow. Mini prepped 2 6 7 9 12 16. Made overnight assay cultures of 3k3-XylE and C3-xylE

Weds 6th Oct

  • Digested mini preps 2 6 7 9 12 16 with E+S will run on gel to determine if it's the correct plasmid, then will select a colony to midi prep tomorrow.
  • Will perform the same assays already performed by Nick with the 3k3-XylE constructs to compare results.
  • Just realised Im working with background colony 16!! disregard this after the gel run!
  • I'm currently keeping my undigested pVeg-XylE minis in Florian's DNA box (orange lids and labels)


Thursday 7th Oct

  • Labpartner, goodmorning and from greece!! :p
  • Niiiiiiiiiiiiiiick!!! you left the country! you were too ugly. good luck babe!
  • Midi prepped pVeg-XylE-B0014 in PSB1C3; will digest out the insert (E+S) and put it into 3K3(E+X). Then both the midi (in PSB1C3) and the ligation 3k3-pVeg-XylE-B0014 can be transformed into TOP10. J23101-XylE-B0014 in 3K3 Vector also needs to be put into TOP10 currently only the PSB1C3 version is in TOP10. We need the rest for comparison testing as TOP10 is the more widely used testing strain.
  • Florian kindly digested pVeg-XylE-B0014-C3 with E+S
  • Ran PCR to get out blunt pVeg-XylE-B0014 using primers. Did 3 reactions at variations around 60 degrees.

Friday 8th Oct

  • Ran gel analysis of my PCRs 1, 2 and 3* appear to have worked. Samples 1 and 3* show stronger bands and so should be used in the next ligation step into the SPEC vector (which kyasha will be doing) then transformation into Bacillus (meeee.)
  • Chris is purifying digested pVeg-XylE-B0014-C3 to get the insert out and this will then be ran on a gel alongside cut (E+X) 3k3 to determine a ratio for ligation.
  • Meeting today at 4pm, no one is here!! it'll be me Florian, Ben and Piotr attending..yikes

Sunday 10th of the 10th of the 2010!!!!! and im in LAB

  • I did ligations of pVeg-XylE into 3k3 and of Reverse-XylE into the digested C-Tev LacI vector.
  • I also set up assay cultures for monday 2x pVegXE 2x XE-C3 2x XE-3K3 and 2x GFP-XylE!!!
Week 15

Monday 11th Oct

  • No Nicolas Kylilis, what a douche!
  • Diluted my overnights 250μl into 5ml to grow up to around 0.5 OD
  • Gonna perform assays with them this afternoon after some growing time :)))))))
  • Doing data analysis with my last assay results comparing high copy (1C3) vs low copy (3K3)and the effects on XylE activity..boring
  • Finalising the polo-shirt logo design, it's looking fiiiine, we're gonna look goooood ;)

Tues 12th Oct

  • Transformations worked! we now have pVeg-XylE in 3k3 and reverse-XylE with lacI promoter (C-TEV vector)i did replica plates, catechol assay plates and overnights for minis. Will mini prep tmro and transform them into TOP10 and we should be good to go with all constructs.
  • Assays messed up, my fault :( 3k3 didnt respond to catechol, so i think that I picked a background colony, i wont pick it again. The results are a bit ridiculous and will probably be chucked.

Weds 13th Oct mini prepped 3k3-pVeg-XylE-B0014 and went to the school workshop! set off overnight midi of culture 9

Thurs 14th Oct Midi prepped 3k3-pVeg

Fri 15th Transformed all my constructs into T0P10 3k3-J23101/pveg and PSB1C3-J23101/pVeg

Output Photo Gallery
First Successful catechol assay :)))
First successful catechol assay
Experiment which determined the yellow product to be exported out of the cells into the media and not retained by the cells
Cell vs supernatant assay
Cuvette assay set up
:-)
Miaow.jpg
Miaow2.jpg
final batch of transformed XylE constructs into TOP10s
Fusion GFP-XylE vs XylE cuvette response assay
View from above
Plate after assay!