Team:Macquarie Australia/trial

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=Tuesday 07/08/12=
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===[[Team:Macquarie/Protocols/Making LB agar plates|Making liquid media, plates & buffers]]===
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*'''Liquid LB Media'''
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*'''SOC Solution'''
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*'''SOB Solution'''
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*'''LB Agar Plates'''
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'''Ampicillin LB Agar Plates:''' 31 plates'''
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'''Chloramphenicol LB Agar Plates:''' 33 Plates'''
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'''Kanamyacin LB Agar Plates:''' 32 Plates'''
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To browse through our notebook simply click on the image with the date. It will expand with what we did that week. To continue on simply click on the next one.</p>
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<div class="accordionButton" id="open"><div id="protocol">Week 1- Tuesday July 31st</div></div>
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<img style="margin:0 auto;float:none;"src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"></img>
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*'''TB buffer.'''
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<div id="protocolcontent">
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<p> With the break between semesters over, the Macquarie iGEM returned to classes. For us this was the first day of iGEM 2012. We had our first meeting and discussed our project. Dr. Louise Brown and Associate Professor Rob Willows were introduced the team and we were began to determine who would take on certain roles within the team. </p>
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<p> We eagerly began our project, deciding to use the novel approach of Gibson Assemble to produce our optimised genes. We had decided to develop a gene-switch controlled by light. To do this a couple of genes would be needed:
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<ol>
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<li>a bacteriophytochrome</li>
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<li>Heme oxygenase</li>
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</ol>
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<h3>Gibson Assembly Planning</h3>
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<p>The bacteriophytochromes from <i>Deinococcus radiodurans</i> and <i>Agrobacterium tumefaciens</i> were chosen. Over the next week Matt Stclair started to develop the G-blocks by:
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<ol>
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<li>Acquiring the DNA sequence</li>
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<li>Translating into the protein sequence </li>
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<li>Using the DNA sequence, optimise codon usage for <i>E. coli</i></li>
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<li>Translating into protein sequence and ensuring no changes in amino acids relative to the original sequence </li>
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</ol></p>
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</div>
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<div class="accordionButton"><div id="protocol">Week 2- Tuesday 7th August</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p>Our lab work began today with the preparation of liquid media, plates and buffers. The protocols we followed are located <a href="https://2012.igem.org/Team:Macquarie/Protocols/Making_LB_agar_plates">here</a>.</p>
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<p>We prepared the following:
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<blockquote> <ul><li>Liquid LB Media
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</li><li>SOC Solution
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</li><li>SOB Solution
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</li><li>LB Agar Plates
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</li><li>Ampicillin LB Agar Plates: 31 plates
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</li><li>Chloramphenicol LB Agar Plates: 33 Plates
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</li><li>Kanamyacin LB Agar Plates: 32 Plates
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</li><li>TB buffer.
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</li><li>TAE buffer.
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</li><li>EDTA buffer.</li></ul> </blockquote>
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<p>While the lab group produced the necessary materials, another team began developing the G Blocks. The G Block team consisted of Ellaina, Matt Smith, Matt Stclair, Harry, Gazelle, Silas, Kim, and Miguel. Matt Stclair spearheaded the effort and proofread all the sequences we had developed to ensure that we had not changed the protein sequence or introduced restriction sites.</p>
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<p>Ryan volunteered to be the wiki chief with Erin helping out throughout the project. Ellaina ensured the key safety questions were answered, and relayed these to the rest of the team. Rob and Sarah took control of the seeking finances to get the team to Hong Kong.</p>
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<p>In a Macquarie iGEM first, the team took a big interest in the idea of human outreach and we had a brainstorming session. Ellaina proposed that we visit a high school while Elle suggested that the Universities open day would be a great opportunity to communicate with the wider community. Ellaina took control of developing our high school visit and Elle took control of Open Day. </p>
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<div class="accordionButton"><div id="protocol">Week 3- Tuesday 14th August</div></div>
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p>In anticipation of our G Blocks arriving next week, competent cells were prepared. The protocol followed can be seen <a href=https://2012.igem.org/Team:Macquarie_Australia/Protocols/Making_competent_Cells">here</a>.</p>
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<p>Human Practice planning went into overdrive this week. To engage the wider community, fluorescent and bioluminescent parts from the registry were selected and transformed in <i>E. coli</i>. Red, Green, Orange, and Cyan Florescent protein as well as Luciferase were all located and transformed in the competent cells available. More details can be seen <a href="https://2012.igem.org/Team:Macquarie_Australia/Protocols/Outreach_Planning">here</a>. </p>
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*'''TAE buffer.'''
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<div class="accordionButton"><div id="protocol">Week 3- Wednesday 15th August</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p>Our open day preparation did not go as planned. Most of our transformations were unsuccessful, we believe this was because SOC was added after the heat shock. As disappointing as this was, there was one little piece of good news.</p>
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<p>A single colony of Top 10 <i>E. coli</i> containing GFP insert was identified on a plate of LB agar media.
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LB agar and nutrient agar plates were struck using this colony. The colony was also used to inoculate 5mL of nutrient broth. Plates and stock were then incubated at 37°C overnight. Both of the streaked plates and the broth showed significant growth of <i>E. coli</i> colonies which could fluoresce when exposed to UV light.</p>
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<p>We got our first news on the finances front. Unfortunately, Qantas declined to sponsor the effort of getting our team to Hong Kong. This was soon forgotten when the Macquarie University School of Medicine, Agilent and the Defence Science Institute (thanks Yagiz) all agreed to sponsor our team. Hong Kong here we come.</p>
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<p>Design of the G Blocks continued. The fragments were designed with melting temperatures <b>NEED TO ASK STCLAIR</b>. However, IDT had significant synthesising them due to GC rich regions. Unfortunately for the MQ iGEM team, IDT is based in the USA and our lab sessions do not coincide with business hours. The G Block saga continued for a couple of weeks with new GC rich regions that affected synthesis identified with each modification made.</p>
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</div>
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<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
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</div>
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*'''EDTA buffer.'''
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<div class="accordionButton"><div id="protocol">Week 4- Tuesday 21st August</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p>The team received the best news of the iGEM experience so far. Our G Blocks finally had no GC rich regions that inhibited synthesis. With the G Blocks arriving next prepared for the assembly and took the opportunity to spend more time planning our two aspects of human practice.</p>
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<p>The <a href="https://2012.igem.org/Team:Macquarie_Australia/Protocols/CompetentCellTesting">efficiency of the competent cells</a> produced was determined by transforming with the fluorescent constructs being used for the open day activities.
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==='''[[Team:Macquarie/Protocols/Designing Gibson Assembly Fragments|Designing Gibson Assembly Fragments]]'''===
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<div class="accordionButton"><div id="protocol">Week 5- Tuesday 28th August</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p> This is a trial. I do not know if this is going to work or not!</p>
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</div>
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<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
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</div>
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*'''Haemoxygenase Gene'''
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<div class="accordionButton"><div id="protocol">Week 5- Friday 31st August</div></div>
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*''' ''Deinococcus radiodurans'' Bacteriophytochrome Gene'''
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<div class="accordionContent">
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*''' ''Agrobacterium tumefaciens'' Bacteriophytochrome Gene '''
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p> This is a trial. I do not know if this is going to work or not!</p>
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</div>
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<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
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<div class="accordionButton"><div id="protocol">Week 6- Tuesday 4th September</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p> This is a trial. I do not know if this is going to work or not!</p>
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</div>
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<div class="accordionButton"><div id="protocol">Week 5- Friday 31st August</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p> This is a trial. I do not know if this is going to work or not!</p>
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</div>
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<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
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</div>
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<div class="accordionButton"><div id="protocol">Week 6- Tuesday 4th September</div></div>
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<div class="accordionContent">
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<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
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<div id="protocolcontent">
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<p> This is a trial. I do not know if this is going to work or not!</p>
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</div>
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----
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=Tuesday 14/8/12=
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[[File:pipettes.jpg|400px|left]]
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(Click on headings to visit methods)
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===[[Team:Macquarie_Australia/Protocols/Making competent Cells|Making Competent Cells]]===
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*'''Choosing Biobricks'''
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*'''Making competent cells'''
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*'''[[Transformation Protocol]]'''
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===[[Team:Macquarie_Australia/Protocols/Outreach Planning |Outreach Planning Labwork ]]===
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 +
*'''Open Day'''
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*'''Secondary Education Seminars'''
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 +
----
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 +
=Wednesday 15/8/12=
 +
Our open day preparation did not go as planned. Most of our transformations were unsuccessful, we believe this was because SOC was added after the heat shock.  As disappointing as this was, there was one little piece of good news.
 +
 
 +
A single colony of Top 10 E.coli containing GFP insert was identified on a plate of LB agar media.
 +
 
 +
LB agar and nutrient agar plates were struck using this colony.  The colony was also used to inoculate 5mL of nutrient broth.  Plates and stock were then incubated at 37°C overnight. Both of the streaked plates and the broth showed significant growth of E.coli colonies which could fluoresce when exposed to UV light.
 +
 
 +
Unfortunately, Qantas declined to sponsor the effort of getting our team to Hong Kong.  This was soon forgotten when the Macquarie University School of Medicine, Agilent and the Defence Science Institute (thanks Yagiz) both agreed to sponsor our team.  Hong Kong here we come.
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 +
 
 +
----
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=Tuesday 21/8/12=
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===[[Team:Macquarie_Australia/Protocols/Outreach Planning Part Two |Outreach Planning Labwork ]]===
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*'''Secondary education seminars'''
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*'''Reattempting glowing bacteria'''
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===[[Team:Macquarie_Australia/Protocols/Making Plates |Making Plates ]]===
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----
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=Tuesday 28/8/12=
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===[[Team:Macquarie_Australia/Protocols/OutreachandSchoolPlanning |Outreach Planning]]===
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*'''High School Presentations: Preparation for 4th September'''
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*'''Open Day: Preparation for 8th September at Macquarie University'''
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*'''Writing the Abstract for the project due 7th September
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===[[Team:Macquarie_Australia/Protocols/Labwork | Outreach Lab Work]]===
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*'''Fluorescent Bacteria'''
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===[[Team:Macquarie_Australia/Protocols/CompetentCellTesting | Competent Cell Testing]]===
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----
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=Friday 31/8/12=
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===[[Team:Macquarie_Australia/Protocols/ArrivalofGBlocks | gBlock fragments]]===
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*'''gBlock fragments from IDT'''
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 +
----
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=Tuesday 04/09/12=
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===[[Team:Macquarie_Australia/Protocols/GibsonAssembly | Gibson Assembly]]===
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*'''Gibson Assembly Protocol'''
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===[[Team: Macquarie_Australia/Protocols/SchoolVisit | Outreach ]]===
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*'''Visit to High School'''
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*'''Outreach administration'''
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*'''Media preparation for more Fluorescent bacteria
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===[https://2012.igem.org/Team:Macquarie_Australia/Protocols/Making_competent_Cells Competent Cell Protocol]===
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*'''Generation of Competent Cells'''
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----
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=Wednesday 05/09/12=
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Matt looked at the transformed cells we cultured the previous day (04/09/12). Two small colonies of the transformed Deinococcus bacteriophytochome without the T7 promoter were grown on kanamycin. The positive control was successful, so we could infer that the reactions were proceeding as expected. The positive control was also cultured on XGal and incubated overnight.
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The Deinococcus was cultured in 2 tubes of LB broth (2 mL) containing kanamycin. The transformations performed on the previous day were done again with a slightly different method. We choose to redo the transformations as the efficiency was relatively low. The transformations were re-innoculated onto fresh plates and incubated over night.
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----
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=Thursday 06/09/12=
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We continued on from the Gibson assembly and transformations we performed on the 04/06/12. Daniel did a plasmid prep using a QIAprep Spin Miniprep Kit by Qiagen, for two colonies from Deinococcus.
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===[https://2012.igem.org/Team:Macquarie_Australia/Protocols/PlasmidPrep QIAprep Spin Miniprep Kit by Qiagen]===
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----
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=Friday 07/09/12=
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Plasmid Preparation was continued from Thursday, by Daniel and Harry, using the QIAprep Spin Miniprep Kit by Qiagen. A total of 7 colony cultures were used as the remaining did not grow. Successful cultures included 6 colonies from  1C  and 1 colony from 3K. The DNA concentration was then determined for each sample by NanoDrop, with EB buffer from the Miniprep Kit used as a blank.
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===[https://2012.igem.org/Team:Macquarie_Australia/Protocols/NanoDropResults NanoDrop Results]===
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===[https://2012.igem.org/Team:Macquarie_Australia/Protocols/SequencingResults Sequencing Results]===
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Purified Plasmid Samples then underwent sequencing at the Macquarie University Sequencing Facility. Each sample contained 10 uL Plasmid (>50ng/ul) and 1 uL of BioBrick forward and reverse sequencing primers.
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----
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=Tuesday 11/09/12=
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The team received excellent news from the sequences performed on Friday. We had produced our first gene that is suitable for a biobrick! We had successfully produced the T7 promoter containing Heme Oxygenase. We then took this plasmid and inserted into a BL21 strain of E. coli to overproduce the protein.
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We also performed another round of Gibson assembly. As the T7 Heme Oxygenase was successfully produced we decided to focus on the Deinococcus and Agrobacterium bacteriophytochromes.
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===[https://2012.igem.org/Team:Macquarie_Australia/Protocols/ResultsofGibsonsofar110912 Results of Gibson Assembly]===
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*'''Further Gibson Assembly lab work'''
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*'''Transformation'''
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*'''More plate preparation - [https://2012.igem.org/Team:Macquarie/Protocols/Making_LB_agar_plates Kanamycin & Chloramphenicol''']
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----
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=Wednesday 12/09/12=
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===[https://2012.igem.org/Team:Macquarie_Australia/Protocols/Construct_olonies_1209 Colony Count Results]===
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----
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=Monday 17/09/12=
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----
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=Tuesday 18/09/12=
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 +
----
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=Wednesday 19/09/12=
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Plates from the transformations of Top 10 cells with the ligation products were inspected.  No colonies from the 2K-4K ligations grew.  9 colonies from the 1C-3C and single colony from 1C-5C colonies had grown. These colonies were used to inoculate into LB Kanamycin and left overnight for growth.
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Rob and Matt spun down the BL21 colonies that had been transformed with plasmids containing the T7-HO genes (1C) and had been growing overnight after inducing with IPTG in the presence of ALA.  2 of the cultures were not induced but still had ALA added.  These cultures had varying levels of green pigment visible in the pellet after being spun down. One culture was subjected to an additional 10ul of 100mM ALA and left to grow in the refrigerator.  This culture will be left to grow for a further 48-72 hours to assess if there is any increase in pigmentation.  Pellets from the remaining treatments are in the -18 freezer and will be used to run SDS-PAGE.
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The liquid cultures used for the earlier minipreps were used to streak agar plates and these are now in the incubator.  The cultures were then transferred into glycerol stocks and frozen.
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 +
----

Latest revision as of 07:15, 20 September 2012



Contents

Tuesday 07/08/12

(Click on headings to visit methods)

Making liquid media, plates & buffers

  • Liquid LB Media
  • SOC Solution
  • SOB Solution
  • LB Agar Plates

Ampicillin LB Agar Plates: 31 plates

Chloramphenicol LB Agar Plates: 33 Plates

Kanamyacin LB Agar Plates: 32 Plates

  • TB buffer.
  • TAE buffer.
  • EDTA buffer.

Designing Gibson Assembly Fragments

  • Haemoxygenase Gene
  • Deinococcus radiodurans Bacteriophytochrome Gene
  • Agrobacterium tumefaciens Bacteriophytochrome Gene

Tuesday 14/8/12

Pipettes.jpg









(Click on headings to visit methods)

Making Competent Cells

Outreach Planning Labwork

  • Open Day
  • Secondary Education Seminars

Wednesday 15/8/12

Our open day preparation did not go as planned. Most of our transformations were unsuccessful, we believe this was because SOC was added after the heat shock. As disappointing as this was, there was one little piece of good news.

A single colony of Top 10 E.coli containing GFP insert was identified on a plate of LB agar media.

LB agar and nutrient agar plates were struck using this colony. The colony was also used to inoculate 5mL of nutrient broth. Plates and stock were then incubated at 37°C overnight. Both of the streaked plates and the broth showed significant growth of E.coli colonies which could fluoresce when exposed to UV light.

Unfortunately, Qantas declined to sponsor the effort of getting our team to Hong Kong. This was soon forgotten when the Macquarie University School of Medicine, Agilent and the Defence Science Institute (thanks Yagiz) both agreed to sponsor our team. Hong Kong here we come.


Tuesday 21/8/12

Outreach Planning Labwork

  • Secondary education seminars
  • Reattempting glowing bacteria

Making Plates

Tuesday 28/8/12

Outreach Planning

  • High School Presentations: Preparation for 4th September
  • Open Day: Preparation for 8th September at Macquarie University
  • Writing the Abstract for the project due 7th September

Outreach Lab Work

  • Fluorescent Bacteria

Competent Cell Testing

Friday 31/8/12

gBlock fragments

  • gBlock fragments from IDT

Tuesday 04/09/12

Gibson Assembly

  • Gibson Assembly Protocol

Outreach

  • Visit to High School
  • Outreach administration
  • Media preparation for more Fluorescent bacteria

Competent Cell Protocol

  • Generation of Competent Cells

Wednesday 05/09/12

Matt looked at the transformed cells we cultured the previous day (04/09/12). Two small colonies of the transformed Deinococcus bacteriophytochome without the T7 promoter were grown on kanamycin. The positive control was successful, so we could infer that the reactions were proceeding as expected. The positive control was also cultured on XGal and incubated overnight.

The Deinococcus was cultured in 2 tubes of LB broth (2 mL) containing kanamycin. The transformations performed on the previous day were done again with a slightly different method. We choose to redo the transformations as the efficiency was relatively low. The transformations were re-innoculated onto fresh plates and incubated over night.

Thursday 06/09/12

We continued on from the Gibson assembly and transformations we performed on the 04/06/12. Daniel did a plasmid prep using a QIAprep Spin Miniprep Kit by Qiagen, for two colonies from Deinococcus.

QIAprep Spin Miniprep Kit by Qiagen

Friday 07/09/12

Plasmid Preparation was continued from Thursday, by Daniel and Harry, using the QIAprep Spin Miniprep Kit by Qiagen. A total of 7 colony cultures were used as the remaining did not grow. Successful cultures included 6 colonies from 1C and 1 colony from 3K. The DNA concentration was then determined for each sample by NanoDrop, with EB buffer from the Miniprep Kit used as a blank.

NanoDrop Results

Sequencing Results

Purified Plasmid Samples then underwent sequencing at the Macquarie University Sequencing Facility. Each sample contained 10 uL Plasmid (>50ng/ul) and 1 uL of BioBrick forward and reverse sequencing primers.

Tuesday 11/09/12

The team received excellent news from the sequences performed on Friday. We had produced our first gene that is suitable for a biobrick! We had successfully produced the T7 promoter containing Heme Oxygenase. We then took this plasmid and inserted into a BL21 strain of E. coli to overproduce the protein.

We also performed another round of Gibson assembly. As the T7 Heme Oxygenase was successfully produced we decided to focus on the Deinococcus and Agrobacterium bacteriophytochromes.

Results of Gibson Assembly

Wednesday 12/09/12

Colony Count Results

Monday 17/09/12

Tuesday 18/09/12

Wednesday 19/09/12

Plates from the transformations of Top 10 cells with the ligation products were inspected. No colonies from the 2K-4K ligations grew. 9 colonies from the 1C-3C and single colony from 1C-5C colonies had grown. These colonies were used to inoculate into LB Kanamycin and left overnight for growth.

Rob and Matt spun down the BL21 colonies that had been transformed with plasmids containing the T7-HO genes (1C) and had been growing overnight after inducing with IPTG in the presence of ALA. 2 of the cultures were not induced but still had ALA added. These cultures had varying levels of green pigment visible in the pellet after being spun down. One culture was subjected to an additional 10ul of 100mM ALA and left to grow in the refrigerator. This culture will be left to grow for a further 48-72 hours to assess if there is any increase in pigmentation. Pellets from the remaining treatments are in the -18 freezer and will be used to run SDS-PAGE.

The liquid cultures used for the earlier minipreps were used to streak agar plates and these are now in the incubator. The cultures were then transferred into glycerol stocks and frozen.


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