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<p>All aspects of our lab work were documented using Benchling, and we have displayed the relevant information on this page.</p>

<p>Click on the buttons below to toggle the lab notes for the respective sections.</p>

<div onclick="displayAllCats()">All</div>

<div onclick="displayCloning()">Cloning</div>

<div onclick="displayProtein()">Protein</div>

<div onclick="displayAssays()">Enzyme Assays</div>

<div onclick="displayPlants()">Plants</div>

<div onclick="displayParts()">Parts</div>

<div onclick="displayInterlab()">Interlab</div>

<div id="week1" class="cloning-h">

<ul>

<li>Luria Broth (LB) media was made and antibiotic plates were poured out (ampicillin and kanamycin).</li>

 

<li>A small scale grow up of NEB Turbo colonies was prepared by Dr Daniel Lorenz Winter.  The cultures were miniprepped and the concentrations of the plasmids were recorded.</li>

 

<li>Homemade Gibson Mastermix was made.</li>

</ul>

<div id="week2" class="cloning-h">

<ul>

<li>pETDuet-1 and pRSFDUet-1 plasmids were PCR linearised and a DpnI digest was performed. An agarose el was ran to check band sizes.</li>

</ul>

            </div>

<div id="week3" class="cloning-h">

<ul>

<li>Gibson cloning was performed on aPFD, aPFD-SpyC, bPFD, bPFD-SnpC, IaaM-SnpT, IaaH-SpyT and mVenus-SpyT. Recombinant plasmids were transformed into cells, colony PCR (cPCR) was performed and the corresponding colonies were grown and miniprepped.</li>

<li>Gel was unsuccessful.</li>

</ul>

<div id="week4" class="cloning-h">

<ul>

<li>cPCR of 5 more colonies were performed on the plates from Week 3.</li>

<li>Gibson Assembly was performed again on the 8 constructs, cPCR was performed and agarose gel was ran. Successful transformants were grown up.</li>

</ul>

<div id="week5" class="cloning-h">

<ul>

<li>Overnight cultures of bacteria were miniprepped and DNA concentrations were recorded.</li>

<li>Experiencing issues with cloning.</li>  

<li>Several controls were tested (NEB, homemade and Daniel Winter’s GIbson Assembly Mastermix).</li>

</ul>

<div id="week6" class="cloning-h assays-h">

<ul>

<li>pETDuet-1 and pRSFDuet-1 plasmids were PCR cleaned up and a nanodrop was performed to check DNA concentrations.</li>

<li>Several more experiments were performed to identify the problem with our cloning experiments. Plasmid backbones, mastermix and DNA inserts were tested and results indicated that it may have been an issue with the Gibson Mastermix.</li>

<li>8 control plates were transformed.</li>

<li>cPCR was performed on the 8 plates.</li>

</ul>

<h4>Enzyme Assays</h4>

<ul>

<li>Salkowski Assay was set up to detect the end-product, Indole Acetic Acid.</li>

<li>Reagents and safety documents were prepared.</li>  

                                    <li>The Salkowski assay was undertaken and absorbance values recorded using a 96 well plate reader.  A linear standard curve was generated.</li>

 

</ul>

<div id="week7" class="cloning-h">

<ul>

    <li>Competent DH5-alpha and T7 cells were made.</li>

</ul>

<div id="week8" class="cloning-h assays-h">

<ul>

<li>IaaH-SpyT and IaaM-SnpT were cloned into pRSFDuet-1 plasmids and transformed into competent cells.</li>  

</ul>

<h4>Enzyme Assay</h4>

<ul>

<li>A standard curve for the Salkowski assay  was produced with the  microtitre plate (250uL volume). The Salkowski reagent and the IaaH stock was used at a 1:2 ratio.</li>  

</ul>

<div id="week9" class="cloning-h">

<ul>

<li>cPCR was performed on the two plates (48 reactions). Colonies were grown up (however due to a mix up of gel images, the wrong IaaH-SpyT colonies were grown up).</li>

<li>Overnight cultures were miniprepped and sent for sequencing. New Gibson Assembly mastermix was made.</li>

<li>aPFD-SpyT and bPFD-SnpC were cloned into pETDuet-1 and transformed into cells.</li>

<li>cPCR was performed on the transformed cells, and small scale grow up was made. Overnight cultures were made and gel was ran on digested products. bPFD-SnpC transformants  were observed, however we were unable to detect presence of aPFD-SpyC insert.</li>

</ul>

<div id="week10" class="cloning-h interlab-h">

<ul>

<li>Linear pETDuet-1 and pRSFDuet-1 plasmid backbones were prepared.</li>

<li>Gibson Assembly was performed on aPFD-SpyC and aPFD again. Transformation was unsuccessful.</li>

<li>IaaM-SnpT plasmids were sent for sequencing.</li>

<li>aPFD, aPFD-SpyC and pET linear/circular controls were transformed into DH5-alpha cells.</li>

<li>Sequencing results for IaaM-Snp were successful.</li>

<li>cPCR (48 reactions ) were performed on the aPFD and aPFD-SpyC plates. Successful colonies were grown up. A miniprep was performed on the overnight cultures, and a double digest was performed to verify presence of the insert by running an agarose gel electrophoresis.</li>

</ul>

<ul>

<li>Chloramphenicol plates were poured out.</li>

</ul>

<ul>

<li>IaaH-SpyT and bPFD-SnpC were cloned into respective plasmid vectors and transformed into cells.</li>  

<li>cPCR was performed (48 reactions) and successful bPFD-SnpC transformants were grown up. None of the bands were at the expected size for the IaaH-SpyT cPCR products.</li>

<li>A miniprep and digest  was performed on bPFD-SnpC, however none of the samples displayed a band at the right size.</li>

</ul>

                <ul>

<li>aPFD and aPFD-SpyC plasmids were transformed into T7 expression cells.</li>

                </ul>

<ul>

<li>Interlab plasmids were transformed into DH5-alpha cells and grown up.</li>  

<li>Calibration assays were performed.</li>

</ul>

<div id="week12" class="cloning-h interlab-h protein-h plants-h">

<ul>

<li>IaaH-SpyT and bPFD-SnpC were cloned into respective plasmids and transformed into cells.</li>

<li>cPCR was performed on and successful bPFD-SnpC transformants were grown up. IaaH-SpyT colonies were unsuccessful.</li>

<li>Overnight cultures of bPFD-Snp were miniprepped and digested. cPCR was performed on 48 colonies from the old IaaH-SpyT plates.</li>

<li>bPFD-SnpC were sent for sequencing. </li>

</ul>

                <ul>

                <li>aPFD and aPFD-SpyC colonies were grown up and lysed with BugBuster reagent. A SDS page gel was ran on the lysed products.</li>

                </ul>

<ul>

<li>½ MS agarose was made and plates were poured out.</li>

<li>IAA solution was made and filter sterilised.</li>

<li>Seeds were sterilised and placed onto the plates with varying concentrations of indole acetic acid (IAA).</li>

<li>Plates were left for incubation</li>

<ul>

<li>DNA from wells 2H, 2L and 2N were transformed again according to iGEM protocol.</li>  

<li>CFU were counted and cell growth assays were performed on the colonies, as well as the positive and negative controls. Absorbances of 1:10 dilution grow ups were measured at 600 nm. </li>

</ul>

<div id="week13" class="cloning-h interlab-h protein-h">

<div onclick="displayDaily13()" class="week-header">

<div class="week-content" id="w13">

                <li>IaaM-SnoopT and bPFD plasmids were transformed into T7 cells. Colonies were grown up with positive and negative controls and the cells were harvested.</li>

<li>2 x grow up of each test device, final OD measurements taken for 1:10 dilution test cultures.</li>

</div>

<div onclick="displayDaily14()" class="week-header">

            <h4>30th July - 5th August</h4>

<div onclick="displayDaily15()" class="week-header">

            <h4>6th August - 12th August</h4>

                <li>IaaM-SnpT, aPFD, bPFD, bPFD-SnpC, aPFD-SpyC  were transformed and expression experiments were attempted. However, the OD of the cultures were too low.</li>

<li>Total cell protein was extracted and an SDS page gel was ran on the products - however there was no expression, including the controls.</li>

<li>Double digested aPFD-SpyC and bPFD-SnoopC cloned into pET-Duet plasmids. Also digested linear pSB1C3 backbone provided by iGEM.</li>

<div class="week-content" id="w16">

<li>mCerulean-SnpT and mVenus-SpyT plasmids were sent for sequencing.</li>

 

 

 

<div class="week-content" id="w17">

<h4>Cloning</h4>

 

 

<li>1L of 1/2 MS (1% sucrose, 1% agar) autoclaved however autoclave experienced error mid-run. Once media was able to be collected it had gone bad.</li>

<div class="week-content" id="w18">

 

 

</div>

 

<div id="week19" class="cloning-h protein-h">

<li>We attempted to clone modified aPFD and bPFD into pETDUet-1. After transformation, cPCR was performed; however we were unable to detect successful transformants.</li>

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

</div>

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