Difference between revisions of "Team:UNSW Australia/Notebook"

Line 42: Line 42:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p>Luria Broth (LB) media was made and antibiotic plates were poured out (ampicillin and kanamycin).</p>
+
<ul>
−
<p>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.</p>
+
<li>Luria Broth (LB) media was made and antibiotic plates were poured out (ampicillin and kanamycin).</li>
−
<p>Homemade Gibson Mastermix was made.</p>
+
  
 +
<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>
 
</div>
 
</div>
 
</div>
Line 58: Line 61:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p>pETDuet-1 and pRSFDUet-1 plasmids were PCR linearised and a DpnI digest was performed. An agarose el was ran to check band sizes.</p>
+
<ul>
−
                        </div>
+
<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>
 
</div>
 
</div>
 
</div>
Line 71: Line 76:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<ul>
+
<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>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>
+
<li>Gel was unsuccessful.</li>
−
</ul>
+
</ul>
−
 
+
 
</div>
 
</div>
 
</div>
 
</div>
Line 88: Line 92:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
 
</div>
Line 101: Line 108:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
 
</div>
Line 114: Line 125:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
  
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assays</h4>
 
<h4>Enzyme Assays</h4>
−
<p></p>
+
<ul>
 +
<li>Salkowski Assay standard curve was generated for Indole acetic acid.</li>
 +
<li>The Salkowski assay was ran within the microtitre plate. And a standardised gradient was produced.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 132: Line 151:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<li>Competent DH5-alpha and T7 cells were made.</li>
 
</div>
 
</div>
 
</div>
 
</div>
Line 145: Line 164:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>IaaH-SpyT and IaaM-SnpT were cloned into pRSFDuet-1 plasmids and transformed into competent cells.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assay</h4>
 
<h4>Enzyme Assay</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
 
</div>
Line 163: Line 186:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div id="week10" class="cloning interlab protein">
+
<div id="week10" class="cloning interlab">
 
<div onclick="displayDaily10()" class="week-header">
 
<div onclick="displayDaily10()" class="week-header">
 
<h3>Week 10</h3>
 
<h3>Week 10</h3>
Line 176: Line 204:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
−
</div>
+
<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>
−
<div class="protein">
+
<li>IaaM-SnpT plasmids were sent for sequencing.</li>
−
<h4>Protein</h4>
+
<li>aPFD, aPFD-SpyC and pET linear/circular controls were transformed into DH5-alpha cells.</li>
−
                <p></p>
+
<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>
 
</div>
 
</div>
  
 
<div class="interlab">
 
<div class="interlab">
 
<h4>Interlab</h4>
 
<h4>Interlab</h4>
−
<p></p>
+
<ul>
 +
<li>Chloramphenicol plates were poured out.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 199: Line 231:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
<li>aPFD and aPFD-SpyC plasmids were transformed into T7 expression cells.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="interlab">
 
<div class="interlab">
 
<h4>Interlab</h4>
 
<h4>Interlab</h4>
−
<p></p>
+
<ul>
 +
<li>Interlab plasmids were transformed into DH5-alpha cells and grown up.</li>
 +
<li>Calibration assays were performed.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 222: Line 263:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <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>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
  
 
<div class="interlab">
 
<div class="interlab">
 
<h4>Interlab</h4>
 
<h4>Interlab</h4>
−
<p></p>
+
<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>
 
</div>
 
</div>
 
</div>
Line 250: Line 305:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>pETDuet-1 and pRSFDuet-1 plasmids were linearised.</li>
 +
<li>cPCR was performed on IaaH-SpyT. Unsuccessful PCR so nothing was grown up.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <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>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="interlab">
 
<div class="interlab">
 
<h4>Interlab</h4>
 
<h4>Interlab</h4>
−
<p></p>
+
<ul>
 +
<li>2 x grow up of each test device, final OD measurements taken for 1:10 dilution test cultures.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 273: Line 335:
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
<li>The total cell protein was extracted and products were treated with BugBuster reagent. An SDS-Page gel and Western Blot was ran, however no results were obtained from these experiments.  The MES buffer precipitated in the fridge.</li>
 +
                </ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 286: Line 350:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>mVenus-SpyT and mCerulean-SnpT were cloned into pRSFDuet-1 plasmids and transformed into cells.</li>
 +
<li>cPCR was performed on the transformed colonies (24 reactions) and a gel was ran. Successful colonies were grown up.</li>
 +
<li>The overnight mVenus and mCerulean cultures were miniprepped and a digest was performed. Gel was ran to check for the presence of the insert.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <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>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
−
</div>
+
<li>Double digested aPFD-SpyC and bPFD-SnoopC cloned into pET-Duet plasmids. Also digested linear pSB1C3 backbone provided by iGEM.</li>
−
 
+
<li>We attempted to gel extract the digested inserts using a Qiagen kit however the yield was very low so we did not proceed with ligation.</li>
−
<div class="interlab">
+
</ul>
−
<h4>Interlab</h4>
+
−
<p></p>
+
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div id="week16" class="cloning protein plants parts assays">
+
<div id="week16" class="cloning protein plants assays">
 
<div onclick="displayDaily16()" class="week-header">
 
<div onclick="displayDaily16()" class="week-header">
 
<h3>Week 16</h3>
 
<h3>Week 16</h3>
Line 314: Line 383:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>mCerulean-SnpT and mVenus-SpyT plasmids were sent for sequencing.</li>
 +
<li>Another attempt was made to clone IaaH-SpyT into pRSFDuet-1. Gibson assembly was performed and the products were transformed into cells.</li>
 +
<li>cPCR was performed (24 reactions) and successful transformants were grown up.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
<li>An attempt was made to transform and express mVenus and mCerulean 3. The cultures were lysed with BugBuster and a gel was ran on the products. We were unable to obtain expression from either parts.</li>
 +
<li>The constitutive BioBrick parts with IaaM and IaaH under the same promoter were expressed.</li>
 +
<li>Large scale expressions of aPFD, bPFD and Kinase (control) were purified, however we were unable to obtain any positive results from this experiment.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<ul>
 +
<li>Prepared ½ MS media with 1% sucrose. Surface sterilised <i>Arabidopsis thaliana</i>seeds added to 250ml of media and stored at 4C to stratify for 3 days. Remaining 250ml media prepared with 1% agar, square plates prepared.</li>
 +
</ul>
 
</div>
 
</div>
  
−
<div class="parts">
 
−
<h4>Parts</h4>
 
−
<p></p>
 
−
</div>
 
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assays</h4>
 
<h4>Enzyme Assays</h4>
−
<p></p>
+
<ul>
 +
<li>Another Salkowski standard curve was produced. Dilutions were done in 5% EtOH.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
  
−
<div id="week17" class="cloning protein plants parts">
+
<div id="week17" class="cloning protein plants">
 
<div onclick="displayDaily17()" class="week-header">
 
<div onclick="displayDaily17()" class="week-header">
 
<h3>Week 17</h3>
 
<h3>Week 17</h3>
Line 346: Line 423:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>Overnight cultures were miniprepped and digested. Gel was ran to check for the presence of IaaH-SpyT insert.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>All of the cloned plasmids were transformed into T7 and commercial Lemo cells. Glycerol stocks were made for all the parts, and were grown up and induced with IPTG. There was a lack of difference between induced and non induced cultures.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<ul>
−
</div>
+
<li>Liquid culture removed from fridge, seed left illuminated at room temperature to germinate.</li>
−
 
+
<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="parts">
+
<li>10mM filter sterilised IAA stock prepared.</li>
−
<h4>Parts</h4>
+
</ul>
−
<p></p>
+
 
</div>
 
</div>
 
</div>
 
</div>
Line 373: Line 453:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>IaaH-SpyT plasmids were sent for sequencing.</li>
 +
<li>We experienced difficulties in protein expression, which may be due to the presence of the iGEM restriction sites preventing successful translation.</li>
 +
<li>Original DNA constructs were PCR modified with primers that cut out the iGEM prefix and suffix.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>His-mCerulean3 and His-mVenus constructs from Dr Dominic Glovers lab, His-mCerulean3-SnpT and Iaa BioBrick parts were grown up and expressed, however expression failed. This was reattempted, however results did not change.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assays</h4>
 
<h4>Enzyme Assays</h4>
−
<p></p>
+
<ul>
 +
<li>HPLC standards made - Trp, IAA, IAM.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 396: Line 484:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>All modified DNA constructs were cloned into corresponding plasmids and transformed into cells.</li>
 +
<li>cPCR yielded unsuccessful transformation results.</li>
 +
<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>
 +
<li>Plasmids were PCR linearised according to protocol.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>mVenus and mCerulean3 were successfully expressed and purified.</li>
 +
<li>Another attempt was made to express aPFD, bPFD, mCerulean3 and mVenus, however there was no expression when the products were ran on an SDS page gel.</li>
 +
                </ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 414: Line 510:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>Another attempt was made in cloning aPFD and bPDS into pETDuet-1. No colonies were formed after transformation.</li>
 +
<li>Another attempt was made using commercial cells from NEB, which yielded several colonies on both plates.</li>
 +
<li>cPCR results did not yield positive results.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>The columns were cleaned.</li>
 +
<li>Another attempt was made to express IaaM, aPFD-SpyC and bPFD-SnpC, mVenus (positive control). Unfortunately, expression was not achieved.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
 +
<li>Phusion PCR amplification of aPFD-SpyC, bPFD-SnoopC and IaaM-SnoopT Gblocks (provided by IDT) performed.</li>
 +
<li>However primers were diluted to 100uM rather than 10uM so  the PCR amplification was unsuccessful. Performed PCR amplification of Gblocks again, however extra bands on gel indicated non-specific binding of primers. We decided to generate inserts by restriction enzyme digest of already cloned pET-Duet and pRSF plasmids in future attempts.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 437: Line 543:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>We made a decision to switch plasmid vectors for our cloning experiments, using pET-19b instead of pETDuet-1/pRSFDuet-1. DNA constructs were PCR amplified using a new set of primers, which would allow the inserts to be cloned into the new vector.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
<li>An attempt was made to express aPFD and bPFD, however no expression was achieved.</li>
 +
<li>The constitutive IaaM and IaaH BioBrick was expressed.</li>
 +
<li>Waiting for the DNA constructs to be cloned into pET-19b plasmid vector.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<ul>
 +
<li>As an MS media alternative, water agar plates prepared with boiled tap water (tap water to utilise trace minerals, boiled to remove chlorine) and 1% agar.</li>
 +
<li>Water agar plates were poured out with varying concentrations of IAA and stored at -20 degrees.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
 +
<li>Double digest of aPFD-SpyC, bPFD-SnoopC and IaaM-Snoop T inserts and linearised pSB1C3 backbone vector with EcoRI-HF and PstI. After digest confirmation via an agarose gel, inserts were ligated with the pSB1C3 vector overnight.</li>
 +
<li>Ligations were transformed into DH5a cells. Transformation using our own attempts to make competent cells failed so were repeated with commercial competent cells.</li> <li>Following cPCR, successful ligations were grown up overnight and miniprepped. Following a diagnostic digest and gel, plasmids with the right insert size were sent for sequencing.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 465: Line 583:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>aPFD, bPFD, IaaH-SpyT and IaaM-SnpT were cloned into pET-19b plasmid vector and transformed into cells. cPCR was performed and successful transformants were grown up.</li>
 +
<li>aPFD, bPFD and IaaH-SpyT were successfully cloned into the new vector. </li>
 +
<li>IaaM-SnpT was PCR amplified again, and the products of the PCR were verified on a gel.</li>
 +
<li>The insert was cloned into pET-19b and transformed into cells.</li>
 +
<li>cPCR was performed on the colonies, however the results of the gel was not successful.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>gPFD-SpyC and gPFD with the double SpyCatcher proteins were expressed. gPFD-2xSpyCacher was successfully purified.</li>
 +
<li>aPFD, bPFD, IaaH-SpyT, gPFD-SpyC and the IaaM+IaaH BioBrick proteins were successfully expressed.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<ul>
 +
<li>Sterilised seeds were added to all the plates. Growth was observed over the next couple of weeks.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
 +
<li>Transformed IaaH BioBrick part (plate 7 W19GI) into DH5a.</li>
 +
<li>Double digest of aPFD, bPFD, IaaH-SpyT and linear pSB1C3 backbone, verified on a gel. Digests were ligated and transformed into DH5a cells.</li>
 +
<li>Sequencing results confirmed aPFD-SpyC, bPFD-SnoopC and IaaM-SnoopT were successfully cloned into the pSB1C3 vector.</li>
 +
</ul>
 
</div>
 
</div>
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assays</h4>
 
<h4>Enzyme Assays</h4>
−
<p></p>
+
<ul>
 +
<li>An assay was performed on the cell pellets of IaaM and IaaH cultures and different time points. Unable to produce positive result.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 497: Line 632:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>New Gibson Assembly mastermix was made and IaaM-SnpT cloning was reattempted. The recombinant plasmid was transformed into cells, however no colonies were yielded.</li>
 +
<li>IaaM-SnpT recombinant plasmid was re-transformed into competent commercial cells.</li>
 +
<li>aPFD-SpyC, bPFD-SnpC, IaaH and IaaM,  were to be PCR amplified using new primers for Gibson Assembly cloning into pET-19b.</li>
 +
<li>A gel was ran on the products to verify that the inserts were successfully amplified.</li>
 +
<li>The inserts (along with mVenus and mCerulean 3, kindly supplied by Django) were cloned into pET-19b and transformed into cells.</li>
 +
<li>aPFD-SpyC, bPFD-SnpC, IaaH, IaaM and IaaM-SnpT were successfully transformed onto plates. cPCR was performed, and successful transformants  were grown up.</li>
 +
<li>Miniprep was performed on the overnight cultures and double digested with EcoRI and XbaI restriction enzymes.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>aPFD, bPFD and IaaH-SpyT cells were sonicated and the proteins were buffer exchanged into PBS. However, the proteins aggregated and crashed.</li>
 +
<li>Attempted to produce a Bradfords standard curve, however it was unsuccessful.</li> 
 +
<li>gPFD-SpyC protein was purified and buffer exchanged into PBS at pH8.</li>
 +
<li>Another attempt was made to express the IaaH+IaaM BioBrick.</li>
 +
<li>The Catcher-Tag chemistry experiment was performed and given to Dr Daniel Lorenz Winter for TEM grid staining.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<ul>
 +
<li>Liquid cultures were trialed where the plants were grown in falcon tubes in MS liquid media.</li>
 +
<li>Tubes were prepared with varying concentrations of IAA (at the lower end of the concentration spectrum.</li>
 +
<li>Final growth was recorded a week after.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
 +
<li>Following cPCR of ligation transformants, successful ligations were grown up overnight and miniprepped.</li>
 +
<li>After confirmation with a diagnostic digestion and gel aPFD and bPFD selected for sequencing. IaaH-SpyC ligation unsuccessful. Another double digest of IaaH performed overnight was unsuccessful, performed two more times to obtain gel-verified digested IaaH-SpyC insert. Digest cleaned-up and ligated with linear pSB1C3 backbone. Transformed into DH5a and putative successful ligations chosen by cPCR and grown up  overnight.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assays</h4>
 
<h4>Enzyme Assays</h4>
−
<p></p>
+
<ul>
 +
<li>Another assay was performed on the cell pellets as well as the supernatant. The assay of the supernatant was successful.</li>
 +
<li>Another Salkowski assay was performed on the cell pellets and supernatants.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 530: Line 689:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>A gel was ran to verify the presence of the inserts, and successful recombinant pET-19b plasmids were sent for sequencing. </li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>Daniel performed TEM on the proteins however the staining was unsuccessful.</li>
 +
<li>A BCA assay was performed on aPFD and bPFD to determine protein concentration.</li>
 +
<li>Catcher-Tag experiment was performed with gPFD and IaaH-SpyT.</li>
 +
<li>aPFD-SpyC, bPFD-SnpC, IaaM, IaaH and IaaM-SnpT were expressed and purified.</li>
 +
<li>A BCA assay was performed on all the purified proteins.</li>
 +
<li>Sec assembly was performed.</li>
 +
<li>FRET experiments were performed with the negative controls.</li>
 +
<li>aPFD-SpyC + IaaH-SpyT assembly.</li>
 +
<li>aPDS-SpyC and IaaH-SpyT assembly experiments were performed.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="plants">
 
<div class="plants">
 
<h4>Plants</h4>
 
<h4>Plants</h4>
−
<p></p>
+
<ul>
 +
<li>Final plant measurements were taken.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
 +
<li>Overnight Dh5a <i>E. coli</i> cultures containing putative  pSB1C3 plasmid containing IaaH-SpyT were miniprepped and verified with diagnostic double digest and gel. Subsequently aPFD, bPFD and IaaH-SpyT in pSB1C3 constructs were sent for sequencing. Sequencing results confirmed successful cloning of aPFD, bPFD and IaaH-SpyT into pSB1C3 vector.</li>
 +
<li>All BioBrick parts dried down in iGEM provided submission plate overnight in drying oven, Sealed with foil and sent to iGEM registry. Final parts sent for submission: aPFD, bPFD, aPFD-SpyC, bPFD-SnoopC, IaaH-SpyT, IaaM-SnoopT.</li>
 +
</ul>
 
</div>
 
</div>
−
 
 
<div class="assays">
 
<div class="assays">
 
<h4>Enzyme Assays</h4>
 
<h4>Enzyme Assays</h4>
−
<p></p>
+
<ul>
 +
<li>Attempt to do salkowski with enzyme.</li>
 +
<li>A final attempt was made to perform the Salkowski assay.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
Line 563: Line 741:
 
<div class="cloning">
 
<div class="cloning">
 
<h4>Cloning</h4>
 
<h4>Cloning</h4>
−
<p></p>
+
<ul>
 +
<li>Final gel depicting all 8  digested pETDuet-1 and pRSFDuet-1 plasmids was ran.</li>
 +
</ul>
 
</div>
 
</div>
  
 
<div class="protein">
 
<div class="protein">
 
<h4>Protein</h4>
 
<h4>Protein</h4>
−
                 <p></p>
+
                 <ul>
 +
                <li>Final TEM imaging by Dr Daniel Winter was performed.</li>
 +
                </ul>
 
</div>
 
</div>
  
 
<div class="parts">
 
<div class="parts">
 
<h4>Parts</h4>
 
<h4>Parts</h4>
−
<p></p>
+
<ul>
 +
<li>Double digest of all plasmid samples sent for submission performed and run on final gel to create image for wiki.</li>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
 
</div>
−
 
−
</div>
 
−
 
 
</div>
 
</div>
  

Revision as of 14:00, 16 October 2018

Notebook

All aspects of our lab work were documented using Benchling, and we have also displayed a portion of the relevant information on this page.

Click on the buttons below to toggle the lab notes for the respective sections.

Week 1

30th April - 6th May

Cloning

  • Luria Broth (LB) media was made and antibiotic plates were poured out (ampicillin and kanamycin).
  • 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.
  • Homemade Gibson Mastermix was made.

Week 2

7th May - 13th May

Cloning

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

Week 3

14th May - 20th May

Cloning

  • 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.
  • Gel was unsuccessful.

Week 4

21st May - 27th May

Cloning

  • cPCR of 5 more colonies were performed on the plates from Week 3.
  • Gibson Assembly was performed again on the 8 constructs, cPCR was performed and agarose gel was ran. Successful transformants were grown up.

Week 5

28th May - 4th June

Cloning

  • Overnight cultures of bacteria were miniprepped and DNA concentrations were recorded.
  • Experiencing issues with cloning.
  • Several controls were tested (NEB, homemade and Daniel Winter’s GIbson Assembly Mastermix).

Week 6

5th June - 10th June

Cloning

  • pETDuet-1 and pRSFDuet-1 plasmids were PCR cleaned up and a nanodrop was performed to check DNA concentrations.
  • 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.
  • 8 control plates were transformed.
  • cPCR was performed on the 8 plates.

Enzyme Assays

  • Salkowski Assay standard curve was generated for Indole acetic acid.
  • The Salkowski assay was ran within the microtitre plate. And a standardised gradient was produced.

Week 7

11th June - 17th June

Cloning

  • Competent DH5-alpha and T7 cells were made.

    • Week 8

    18th June - 24th June

    Cloning

    • IaaH-SpyT and IaaM-SnpT were cloned into pRSFDuet-1 plasmids and transformed into competent cells.

    Enzyme Assay

    • 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.

    Week 9

    25th June - 1st July

    Cloning

    • 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).
    • Overnight cultures were miniprepped and sent for sequencing. New Gibson Assembly mastermix was made.
    • aPFD-SpyT and bPFD-SnpC were cloned into pETDuet-1 and transformed into cells.
    • 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.

    Week 10

    2nd July - 8th July

    Cloning

    • Linear pETDuet-1 and pRSFDuet-1 plasmid backbones were prepared.
    • Gibson Assembly was performed on aPFD-SpyC and aPFD again. Transformation was unsuccessful.
    • IaaM-SnpT plasmids were sent for sequencing.
    • aPFD, aPFD-SpyC and pET linear/circular controls were transformed into DH5-alpha cells.
    • Sequencing results for IaaM-Snp were successful.
    • 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.

    Interlab

    • Chloramphenicol plates were poured out.

    Week 11

    9th July - 15th July

    Cloning

      IaaH-SpyT and bPFD-SnpC were cloned into respective plasmid vectors and transformed into cells.
    • 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.
    • A miniprep and digest was performed on bPFD-SnpC, however none of the samples displayed a band at the right size.

    Protein

    • aPFD and aPFD-SpyC plasmids were transformed into T7 expression cells.

    Interlab

    • Interlab plasmids were transformed into DH5-alpha cells and grown up.
    • Calibration assays were performed.

    Week 12

    16th July - 22nd July

    Cloning

    • IaaH-SpyT and bPFD-SnpC were cloned into respective plasmids and transformed into cells.
    • cPCR was performed on and successful bPFD-SnpC transformants were grown up. IaaH-SpyT colonies were unsuccessful.
    • Overnight cultures of bPFD-Snp were miniprepped and digested. cPCR was performed on 48 colonies from the old IaaH-SpyT plates.
    • bPFD-SnpC were sent for sequencing.

    Protein

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

    Plants

    • ½ MS agarose was made and plates were poured out.
    • IAA solution was made and filter sterilised.
    • Seeds were sterilised and placed onto the plates with varying concentrations of indole acetic acid (IAA).
    • Plates were left for incubation

    Interlab

    • DNA from wells 2H, 2L and 2N were transformed again according to iGEM protocol.
    • 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.

    Week 13

    23rd July - 29th July

    Cloning

    • pETDuet-1 and pRSFDuet-1 plasmids were linearised.
    • cPCR was performed on IaaH-SpyT. Unsuccessful PCR so nothing was grown up.

    Protein

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

    Interlab

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

    Week 14

    30th July - 5th August

    Protein

    • The total cell protein was extracted and products were treated with BugBuster reagent. An SDS-Page gel and Western Blot was ran, however no results were obtained from these experiments. The MES buffer precipitated in the fridge.

    Week 15

    6th August - 12th August

    Cloning

    • mVenus-SpyT and mCerulean-SnpT were cloned into pRSFDuet-1 plasmids and transformed into cells.
    • cPCR was performed on the transformed colonies (24 reactions) and a gel was ran. Successful colonies were grown up.
    • The overnight mVenus and mCerulean cultures were miniprepped and a digest was performed. Gel was ran to check for the presence of the insert.

    Protein

    • IaaM-SnpT, aPFD, bPFD, bPFD-SnpC, aPFD-SpyC were transformed and expression experiments were attempted. However, the OD of the cultures were too low.
    • Total cell protein was extracted and an SDS page gel was ran on the products - however there was no expression, including the controls.

    Parts

    • Double digested aPFD-SpyC and bPFD-SnoopC cloned into pET-Duet plasmids. Also digested linear pSB1C3 backbone provided by iGEM.
    • We attempted to gel extract the digested inserts using a Qiagen kit however the yield was very low so we did not proceed with ligation.

    Week 16

    13th August - 19th August

    Cloning

    • mCerulean-SnpT and mVenus-SpyT plasmids were sent for sequencing.
    • Another attempt was made to clone IaaH-SpyT into pRSFDuet-1. Gibson assembly was performed and the products were transformed into cells.
    • cPCR was performed (24 reactions) and successful transformants were grown up.

    Protein

    • An attempt was made to transform and express mVenus and mCerulean 3. The cultures were lysed with BugBuster and a gel was ran on the products. We were unable to obtain expression from either parts.
    • The constitutive BioBrick parts with IaaM and IaaH under the same promoter were expressed.
    • Large scale expressions of aPFD, bPFD and Kinase (control) were purified, however we were unable to obtain any positive results from this experiment.

    Plants

    • Prepared ½ MS media with 1% sucrose. Surface sterilised Arabidopsis thalianaseeds added to 250ml of media and stored at 4C to stratify for 3 days. Remaining 250ml media prepared with 1% agar, square plates prepared.

    Enzyme Assays

    • Another Salkowski standard curve was produced. Dilutions were done in 5% EtOH.

    Week 17

    20th August - 26th August

    Cloning

    • Overnight cultures were miniprepped and digested. Gel was ran to check for the presence of IaaH-SpyT insert.

    Protein

    • All of the cloned plasmids were transformed into T7 and commercial Lemo cells. Glycerol stocks were made for all the parts, and were grown up and induced with IPTG. There was a lack of difference between induced and non induced cultures.

    Plants

    • Liquid culture removed from fridge, seed left illuminated at room temperature to germinate.
    • 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.
    • 10mM filter sterilised IAA stock prepared.

    Week 18

    27th August - 2nd September

    Cloning

    • IaaH-SpyT plasmids were sent for sequencing.
    • We experienced difficulties in protein expression, which may be due to the presence of the iGEM restriction sites preventing successful translation.
    • Original DNA constructs were PCR modified with primers that cut out the iGEM prefix and suffix.

    Protein

    • His-mCerulean3 and His-mVenus constructs from Dr Dominic Glovers lab, His-mCerulean3-SnpT and Iaa BioBrick parts were grown up and expressed, however expression failed. This was reattempted, however results did not change.

    Enzyme Assays

    • HPLC standards made - Trp, IAA, IAM.

    Week 19

    3rd September - 9th September

    Cloning

    • All modified DNA constructs were cloned into corresponding plasmids and transformed into cells.
    • cPCR yielded unsuccessful transformation results.
    • We attempted to clone modified aPFD and bPFD into pETDUet-1. After transformation, cPCR was performed; however we were unable to detect successful transformants.
    • Plasmids were PCR linearised according to protocol.

    Protein

    • mVenus and mCerulean3 were successfully expressed and purified.
    • Another attempt was made to express aPFD, bPFD, mCerulean3 and mVenus, however there was no expression when the products were ran on an SDS page gel.

    Week 20

    10th September - 16th September

    Cloning

    • Another attempt was made in cloning aPFD and bPDS into pETDuet-1. No colonies were formed after transformation.
    • Another attempt was made using commercial cells from NEB, which yielded several colonies on both plates.
    • cPCR results did not yield positive results.

    Protein

    • The columns were cleaned.
    • Another attempt was made to express IaaM, aPFD-SpyC and bPFD-SnpC, mVenus (positive control). Unfortunately, expression was not achieved.

    Parts

    • Phusion PCR amplification of aPFD-SpyC, bPFD-SnoopC and IaaM-SnoopT Gblocks (provided by IDT) performed.
    • However primers were diluted to 100uM rather than 10uM so the PCR amplification was unsuccessful. Performed PCR amplification of Gblocks again, however extra bands on gel indicated non-specific binding of primers. We decided to generate inserts by restriction enzyme digest of already cloned pET-Duet and pRSF plasmids in future attempts.

    Week 21

    17th September - 23rd September

    Cloning

    • We made a decision to switch plasmid vectors for our cloning experiments, using pET-19b instead of pETDuet-1/pRSFDuet-1. DNA constructs were PCR amplified using a new set of primers, which would allow the inserts to be cloned into the new vector.

    Protein

    • An attempt was made to express aPFD and bPFD, however no expression was achieved.
    • The constitutive IaaM and IaaH BioBrick was expressed.
    • Waiting for the DNA constructs to be cloned into pET-19b plasmid vector.

    Plants

    • As an MS media alternative, water agar plates prepared with boiled tap water (tap water to utilise trace minerals, boiled to remove chlorine) and 1% agar.
    • Water agar plates were poured out with varying concentrations of IAA and stored at -20 degrees.

    Parts

    • Double digest of aPFD-SpyC, bPFD-SnoopC and IaaM-Snoop T inserts and linearised pSB1C3 backbone vector with EcoRI-HF and PstI. After digest confirmation via an agarose gel, inserts were ligated with the pSB1C3 vector overnight.
    • Ligations were transformed into DH5a cells. Transformation using our own attempts to make competent cells failed so were repeated with commercial competent cells.
    • Following cPCR, successful ligations were grown up overnight and miniprepped. Following a diagnostic digest and gel, plasmids with the right insert size were sent for sequencing.

    Week 22

    24th September - 30th September

    Cloning

    • aPFD, bPFD, IaaH-SpyT and IaaM-SnpT were cloned into pET-19b plasmid vector and transformed into cells. cPCR was performed and successful transformants were grown up.
    • aPFD, bPFD and IaaH-SpyT were successfully cloned into the new vector.
    • IaaM-SnpT was PCR amplified again, and the products of the PCR were verified on a gel.
    • The insert was cloned into pET-19b and transformed into cells.
    • cPCR was performed on the colonies, however the results of the gel was not successful.

    Protein

    • gPFD-SpyC and gPFD with the double SpyCatcher proteins were expressed. gPFD-2xSpyCacher was successfully purified.
    • aPFD, bPFD, IaaH-SpyT, gPFD-SpyC and the IaaM+IaaH BioBrick proteins were successfully expressed.

    Plants

    • Sterilised seeds were added to all the plates. Growth was observed over the next couple of weeks.

    Parts

    • Transformed IaaH BioBrick part (plate 7 W19GI) into DH5a.
    • Double digest of aPFD, bPFD, IaaH-SpyT and linear pSB1C3 backbone, verified on a gel. Digests were ligated and transformed into DH5a cells.
    • Sequencing results confirmed aPFD-SpyC, bPFD-SnoopC and IaaM-SnoopT were successfully cloned into the pSB1C3 vector.

    Enzyme Assays

    • An assay was performed on the cell pellets of IaaM and IaaH cultures and different time points. Unable to produce positive result.

    Week 23

    1st October - 6th October

    Cloning

    • New Gibson Assembly mastermix was made and IaaM-SnpT cloning was reattempted. The recombinant plasmid was transformed into cells, however no colonies were yielded.
    • IaaM-SnpT recombinant plasmid was re-transformed into competent commercial cells.
    • aPFD-SpyC, bPFD-SnpC, IaaH and IaaM, were to be PCR amplified using new primers for Gibson Assembly cloning into pET-19b.
    • A gel was ran on the products to verify that the inserts were successfully amplified.
    • The inserts (along with mVenus and mCerulean 3, kindly supplied by Django) were cloned into pET-19b and transformed into cells.
    • aPFD-SpyC, bPFD-SnpC, IaaH, IaaM and IaaM-SnpT were successfully transformed onto plates. cPCR was performed, and successful transformants were grown up.
    • Miniprep was performed on the overnight cultures and double digested with EcoRI and XbaI restriction enzymes.

    Protein

    • aPFD, bPFD and IaaH-SpyT cells were sonicated and the proteins were buffer exchanged into PBS. However, the proteins aggregated and crashed.
    • Attempted to produce a Bradfords standard curve, however it was unsuccessful.
    • gPFD-SpyC protein was purified and buffer exchanged into PBS at pH8.
    • Another attempt was made to express the IaaH+IaaM BioBrick.
    • The Catcher-Tag chemistry experiment was performed and given to Dr Daniel Lorenz Winter for TEM grid staining.

    Plants

    • Liquid cultures were trialed where the plants were grown in falcon tubes in MS liquid media.
    • Tubes were prepared with varying concentrations of IAA (at the lower end of the concentration spectrum.
    • Final growth was recorded a week after.

    Parts

    • Following cPCR of ligation transformants, successful ligations were grown up overnight and miniprepped.
    • After confirmation with a diagnostic digestion and gel aPFD and bPFD selected for sequencing. IaaH-SpyC ligation unsuccessful. Another double digest of IaaH performed overnight was unsuccessful, performed two more times to obtain gel-verified digested IaaH-SpyC insert. Digest cleaned-up and ligated with linear pSB1C3 backbone. Transformed into DH5a and putative successful ligations chosen by cPCR and grown up overnight.

    Enzyme Assays

    • Another assay was performed on the cell pellets as well as the supernatant. The assay of the supernatant was successful.
    • Another Salkowski assay was performed on the cell pellets and supernatants.

    Week 24

    8th October - 14th October

    Cloning

    • A gel was ran to verify the presence of the inserts, and successful recombinant pET-19b plasmids were sent for sequencing.

    Protein

    • Daniel performed TEM on the proteins however the staining was unsuccessful.
    • A BCA assay was performed on aPFD and bPFD to determine protein concentration.
    • Catcher-Tag experiment was performed with gPFD and IaaH-SpyT.
    • aPFD-SpyC, bPFD-SnpC, IaaM, IaaH and IaaM-SnpT were expressed and purified.
    • A BCA assay was performed on all the purified proteins.
    • Sec assembly was performed.
    • FRET experiments were performed with the negative controls.
    • aPFD-SpyC + IaaH-SpyT assembly.
    • aPDS-SpyC and IaaH-SpyT assembly experiments were performed.

    Plants

    • Final plant measurements were taken.

    Parts

    • Overnight Dh5a E. coli cultures containing putative pSB1C3 plasmid containing IaaH-SpyT were miniprepped and verified with diagnostic double digest and gel. Subsequently aPFD, bPFD and IaaH-SpyT in pSB1C3 constructs were sent for sequencing. Sequencing results confirmed successful cloning of aPFD, bPFD and IaaH-SpyT into pSB1C3 vector.
    • All BioBrick parts dried down in iGEM provided submission plate overnight in drying oven, Sealed with foil and sent to iGEM registry. Final parts sent for submission: aPFD, bPFD, aPFD-SpyC, bPFD-SnoopC, IaaH-SpyT, IaaM-SnoopT.

    Enzyme Assays

    • Attempt to do salkowski with enzyme.
    • A final attempt was made to perform the Salkowski assay.

    Week 25

    15th October - 21st October

    Cloning

    • Final gel depicting all 8 digested pETDuet-1 and pRSFDuet-1 plasmids was ran.

    Protein

    • Final TEM imaging by Dr Daniel Winter was performed.

    Parts

    • Double digest of all plasmid samples sent for submission performed and run on final gel to create image for wiki.