Team:Exeter/lab book/1gp/wk12

From 2012.igem.org

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     <p><u><b>Monday 24th September (9.00)</u></b></p>
     <p><u><b>Monday 24th September (9.00)</u></b></p>
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<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Adding cultures</u></a> with pBAD(weak)+RBS-GFP, pLacI/Ara-1 pLacI/Ara-1+RBS-GFP and pLacI/Ara-1+RBS-WclY-terminator into liquid medium and incubation overnight
+
<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Adding cultures</u></a> with pBAD(weak)+RBS-GFP, pLacI/Ara-1+RBS-GFP and pLacI/Ara-1+RBS-WclY-terminator into liquid medium and incubation overnight
<p> Uninduced and induced (L-arabinose for pBAD(weak) and IPTG for pLacI/Ara-1) tubes for each of the three constructs were added along with chloramphenicol (for pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP) and ampicillin (for pLacI/Ara-1+RBS-WclY-terminator) as the selection antibiotics. pLacI/Ara-1+RBS-WclY-terminator was used to determine basal levels of GFP fluorescence.</p>
<p> Uninduced and induced (L-arabinose for pBAD(weak) and IPTG for pLacI/Ara-1) tubes for each of the three constructs were added along with chloramphenicol (for pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP) and ampicillin (for pLacI/Ara-1+RBS-WclY-terminator) as the selection antibiotics. pLacI/Ara-1+RBS-WclY-terminator was used to determine basal levels of GFP fluorescence.</p>
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<p><u><b>Tuesday 26th September (9.00)</u></b></p>
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<p><u><b>Tuesday 25th September (9.00)</u></b></p>
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<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#1d1d1b"><u>Mini-Prepping</u></a> of OmpA</p>
+
<p>• Measuring GFP fluorescence of pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP
-
<p>• Send off OmpA for sequencing</p>
+
<p> Same method as followed for protein expression on Thursday 20th September, but 6 conical flasks were used for testing of pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP as well as determining basal levels for pLacI/Ara-1+RBS-WclY-terminator. However, 750µL was only transferred to cuvettes for measuring OD600 at 2 and a half hours after putting all 6 conical flasks in the shaking incubator. The results for OD600 measured is as follows:</p>
-
<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>Transformation</u></a> of pBAD/AraC promoter weak+RBS-GFP, pBAD/AraC promoter strong+RBS-GFP, pBAD(large)+RBS-GFP, constitutive promoter+RBS-GFP, pLacI/Ara-1+RBS-GFP, WclY-pSB1C3, WbnJ-pSB1C3, WbbC-pSB1C3, WfcA-terminator and RBS-WbiP </p>
+
<p>o 1a = 0.134.    |    o 1a = 0.922.</p>
 +
<p>o 1b = 0.162.    |    o 1b = 1.368.</p>
 +
<p>o 2a = 0.149.    |    o 2a = 0.965.</p>
 +
<p>o 2b = 0.144.     |    o 2b = 0.958.</p>
 +
<p>o 3a = 0.155.    |    o 3a = 1.041.</p>
 +
<p>o 3b = 0.137.    |    o 3b = 1.009.</p>
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<p><u><b>Wednesday 27th September (9.00)</u></b></p>
+
<p><i> At: 9:45 and 12:15 left to right respectively</p></i>   
 +
<p> ------------------ </p>
 +
<p>1 = pLacI/Ara-1+RBS-WclY-terminator</p>                     
 +
<p>2 = pLacI/Ara-1+RBS-GFP</p>                             
 +
<p>3 = pBAD(weak)+RBS-GFP</p>
 +
<p> ------------------ </p>
 +
<p>a = uninduced (- inducer)</p>
 +
<p>b = induced (+ inducer)</p>
 +
 
 +
 
 +
<p><u><b>Wednesday 26th September (9.00)</u></b></p>
<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#1d1d1b"><u>Mini-Prepping</u></a> of OmpA</p>
<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#1d1d1b"><u>Mini-Prepping</u></a> of OmpA</p>
<p>• Send off OmpA for sequencing</p>
<p>• Send off OmpA for sequencing</p>

Revision as of 16:35, 25 September 2012

ExiGEM2012 Lab Book 1GP wk12

Single Gene Plasmids and Enzyme Characterisation: 24th - 28th September 2012

Monday 24th September (9.00)

• Adding cultures with pBAD(weak)+RBS-GFP, pLacI/Ara-1+RBS-GFP and pLacI/Ara-1+RBS-WclY-terminator into liquid medium and incubation overnight

Uninduced and induced (L-arabinose for pBAD(weak) and IPTG for pLacI/Ara-1) tubes for each of the three constructs were added along with chloramphenicol (for pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP) and ampicillin (for pLacI/Ara-1+RBS-WclY-terminator) as the selection antibiotics. pLacI/Ara-1+RBS-WclY-terminator was used to determine basal levels of GFP fluorescence.

Tuesday 25th September (9.00)

• Measuring GFP fluorescence of pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP

Same method as followed for protein expression on Thursday 20th September, but 6 conical flasks were used for testing of pBAD(weak)+RBS-GFP and pLacI/Ara-1+RBS-GFP as well as determining basal levels for pLacI/Ara-1+RBS-WclY-terminator. However, 750µL was only transferred to cuvettes for measuring OD600 at 2 and a half hours after putting all 6 conical flasks in the shaking incubator. The results for OD600 measured is as follows:

o 1a = 0.134. | o 1a = 0.922.

o 1b = 0.162. | o 1b = 1.368.

o 2a = 0.149. | o 2a = 0.965.

o 2b = 0.144. | o 2b = 0.958.

o 3a = 0.155. | o 3a = 1.041.

o 3b = 0.137. | o 3b = 1.009.

At: 9:45 and 12:15 left to right respectively

------------------

1 = pLacI/Ara-1+RBS-WclY-terminator

2 = pLacI/Ara-1+RBS-GFP

3 = pBAD(weak)+RBS-GFP

------------------

a = uninduced (- inducer)

b = induced (+ inducer)

Wednesday 26th September (9.00)

• Mini-Prepping of OmpA

• Send off OmpA for sequencing

• Transformation of pBAD/AraC promoter weak+RBS-GFP, pBAD/AraC promoter strong+RBS-GFP, pBAD(large)+RBS-GFP, constitutive promoter+RBS-GFP, pLacI/Ara-1+RBS-GFP, WclY-pSB1C3, WbnJ-pSB1C3, WbbC-pSB1C3, WfcA-terminator and RBS-WbiP

Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/1gp/wk12"