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

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                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         Transformed Εscherichia coli DH5α cells with the interlab testing devices.Transformation was performed twice, due to poor colony growth in the LB + cam media at the first round of transformations.
+
                         Transformed <i>Εscherichia coli</i> DH5α cells with the interlab testing devices.Transformation was performed twice, due to poor colony growth in the LB + cam media at the first round of transformations.
 
                     </li>
 
                     </li>
 
                 </ul>
 
                 </ul>
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                 <ul class="normal">
 
                 <ul class="normal">
 
                     <li>
 
                     <li>
−
                         We transformed the DVR1-ChlG and ChlD-ChlI2 ligation samples into competent DH5α E.coli cells. DVR1-ChlG had successful transformants which were then incubated in LB broth + Kan. ChlD-ChlI2 gave an unsuccessful transformation, so the process was repeated successfully this time. The samples were then liquid cultured.
+
                         We transformed the DVR1-ChlG and ChlD-ChlI2 ligation samples into competent DH5α <i>E.coli</i> cells. DVR1-ChlG had successful transformants which were then incubated in LB broth + Kan. ChlD-ChlI2 had given an unsuccessful transformation, so the process was repeated and was successful the second time. The samples were then liquid cultured.
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
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                 <ul class="indented">
 
                 <ul class="indented">
 
                     <li>
 
                     <li>
−
                         The gel showed unsuccessful ligations so we are switched our ligation technique to standard assembly and backbone swapping of necessary parts
+
                         The gel showed unsuccessful ligations so we switched our ligation technique to standard assembly and backbone swapping of necessary parts
 
                     </li>
 
                     </li>
 
                 </ul>
 
                 </ul>
 
                 <ul class="normal">
 
                 <ul class="normal">
 
                     <li>
 
                     <li>
−
                         We backbone swapped DVR1 to Kan and were in the process of swapping ChlD, trc-POR and GUN4
+
                         We backbone-swapped DVR1 to Kan and were in the process of swapping ChlD, trc-POR and GUN4
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
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                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         Sent successful samples of each off for sequencing
+
                         Sent off successful samples of each for sequencing
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
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                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         We then transformed, liquid cultured, miniprepped and screened on a 1% agarose gel
+
                         We then transformed, liquid-cultured, miniprepped and screened on a 1% agarose gel
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         We had successes for trc-ChlI1 in Amp which we sent for sequencing confirmation
+
                         We had successes for trc-ChlI1 in Amp, which we sent for sequencing confirmation
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         Used standard assembly to assemble:
+
                         Proceeded with standard assembly for:
 
                     </li>
 
                     </li>
 
                 </ul>
 
                 </ul>
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                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
−
                         Sequencing came back for trc-ChlH to show that the promoter swap was unsuccessful. A reattempt of promoter swap is now underway
+
                         Sequencing came back for trc-ChlH to show that the promoter swap was unsuccessful. A reattempt of promoter swap was then undertaken
 
                     </li>
 
                     </li>
 
                     <li>
 
                     <li>
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                     <li>Screened trc-ChlI1 and sent 1 successful plasmid away for sequencing</li>
 
                     <li>Screened trc-ChlI1 and sent 1 successful plasmid away for sequencing</li>
 
                     <li>Performed SDS-PAGE comparing trc-FNR-fdx and lac-FNR-fdx</li>
 
                     <li>Performed SDS-PAGE comparing trc-FNR-fdx and lac-FNR-fdx</li>
−
                     <li>Liquid cultured, Miniprep and screened trc-ChlH, trc-POR-ChlP, trc-ycf39-HliD, trc-FNR and trc-FNR-fdx and ChlI2-ChlD </li>
+
                     <li>Liquid-cultured, Miniprepped and screened trc-ChlH, trc-POR-ChlP, trc-ycf39-HliD, trc-FNR and trc-FNR-fdx and ChlI2-ChlD </li>
 
                     <ul class="indented">
 
                     <ul class="indented">
 
                         <li>
 
                         <li>
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             <div class="panel">
 
             <div class="panel">
 
                 <ul class="normal">
 
                 <ul class="normal">
−
                     <li>Transformed, Liquid cultured and miniprepped  trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP</li>
+
                     <li>Transformed, Liquid-cultured and miniprepped  trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP</li>
 
                     <li>Made glycerol stock for trc-POR and trc-ChlH</li>
 
                     <li>Made glycerol stock for trc-POR and trc-ChlH</li>
−
                     <li>Digested and Screened trc-ChlH-GUN4, trc-FNR and trc-FNR-fdx. Only trc-FNR-fdx and trc-ChlH-GUN4 had successes, which were sent off for sequencing</li>
+
                     <li>Digested and screened trc-ChlH-GUN4, trc-FNR and trc-FNR-fdx. Only trc-FNR-fdx and trc-ChlH-GUN4 had successes, which were sent off for sequencing</li>
−
                     <li>Induced trc-POR cells and run the cell lysate on SDS-PAGE</li>
+
                     <li>Induced trc-POR cells and ran the cell lysate on SDS-PAGE</li>
 
                 </ul>
 
                 </ul>
 
             </div>
 
             </div>
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                 <ul class="normal">
 
                 <ul class="normal">
 
                     <li>Digested and screened  trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP on a 1% agarose gel and sent successful samples for sequencing</li>
 
                     <li>Digested and screened  trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP on a 1% agarose gel and sent successful samples for sequencing</li>
−
                     <li>Backbone swapped lac-CTH1-ycf54-ChlM (Kan)
+
                     <li>Backbone-swapped lac-CTH1-ycf54-ChlM (Kan)
−
                     <li>Performed standard assembly on trc-POR-ChlP-DVR1-ChlG and sent successes for sequencing</li>
+
                     <li>Performed standard assembly on trc-POR-ChlP-DVR1-ChlG and sent successful ones for sequencing</li>
 
                     <li>Induced new trc-POR samples and ran cell lysate on an SDS-PAGE </li>
 
                     <li>Induced new trc-POR samples and ran cell lysate on an SDS-PAGE </li>
−
                     <li>Grow barley for protochlorophyllide extraction</li>
+
                     <li>Grew barley for protochlorophyllide extraction</li>
 
                     <li>Made up more plasmid stock of trc-POR</li>
 
                     <li>Made up more plasmid stock of trc-POR</li>
 
                     <li>Made up glycerol stock of ChlI1</li>
 
                     <li>Made up glycerol stock of ChlI1</li>
 
                     <li>Redesigned and ordered sequencing primers</li>
 
                     <li>Redesigned and ordered sequencing primers</li>
 
                     <li>Tried to insert G-block for yidC and trc-FNR into ampicillin backbone, no successes from screened samples</li>
 
                     <li>Tried to insert G-block for yidC and trc-FNR into ampicillin backbone, no successes from screened samples</li>
−
                     <li>Digestion/ Ligation of trc-ChlI1-ChlI2-ChlD + ChlH-GUN4,  Backbone swap of trc-POR (switched to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR</li>
+
                     <li>Digestion/ Ligation of trc-ChlI1-ChlI2-ChlD + ChlH-GUN4. Backbone swap of trc-POR (switched to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR</li>
 
                 </ul>
 
                 </ul>
 
                 <img src="https://static.igem.org/mediawiki/2018/thumb/8/86/T--Macquarie_Australia--Watlab_Week9_1.png/800px-T--Macquarie_Australia--Watlab_Week9_1.png.jpeg" style="width: 100%;">
 
                 <img src="https://static.igem.org/mediawiki/2018/thumb/8/86/T--Macquarie_Australia--Watlab_Week9_1.png/800px-T--Macquarie_Australia--Watlab_Week9_1.png.jpeg" style="width: 100%;">
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                 <ul class="normal">
 
                 <ul class="normal">
 
                     <li>Received sequencing results for trc-POR-ChlP and sent off trc-POR-ChlP-DVR1-ChlG samples for sequencing </li>
 
                     <li>Received sequencing results for trc-POR-ChlP and sent off trc-POR-ChlP-DVR1-ChlG samples for sequencing </li>
−
                     <li>Digested, Ligated, transformed and liquid cultured lac-CTH1-ycf54-ChlM + trc-FNR-fdx</li>
+
                     <li>Digested, ligated, transformed and liquid-cultured lac-CTH1-ycf54-ChlM + trc-FNR-fdx</li>
−
                     <li>Transformed trc-ChlI1-ChlI2-ChlD + trc-ChlH-GUN4 (shortened to trc-IH),  Backbone swap of trc-POR (to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR</li>
+
                     <li>Transformed trc-ChlI1-ChlI2-ChlD + trc-ChlH-GUN4 (shortened to trc-IH),  Backbone-swapped trc-POR (to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
−
                         <li>Liquid Cultured, miniprepped and single/double screening digests on a 1% agarose gel</li>
+
                         <li>Liquid-cultured, miniprepped and performed single/double screening of digests on a 1% agarose gel</li>
 
                         <li>Sent 3 IH samples and 1 of both trc-POR (Cam) and lac-CTH1-ycf54-ChlM-trc-POR for sequencing</li>
 
                         <li>Sent 3 IH samples and 1 of both trc-POR (Cam) and lac-CTH1-ycf54-ChlM-trc-POR for sequencing</li>
 
                     </ul>
 
                     </ul>
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             <div class="panel">
 
             <div class="panel">
 
                 <ul class="normal">
 
                 <ul class="normal">
−
                     <li>Received sequencing results for trc-ChlI1-ChlI2-ChlD-ChlH-GUN4, discovered trc-ChlI1 had not ligated initially but all other genes were present and sequence confirmed</li>
+
                     <li>Received sequencing results for trc-ChlI1-ChlI2-ChlD-ChlH-GUN4, discovered trc-ChlI1 had not ligated initially but all other genes were present and sequence was confirmed</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
 
                         <li>Stopped streamline operon and digested and ligated trc-ChlI1 to ChlI2-ChlD-ChlH-GUN4</li>
 
                         <li>Stopped streamline operon and digested and ligated trc-ChlI1 to ChlI2-ChlD-ChlH-GUN4</li>
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             <div class="panel">
 
             <div class="panel">
 
                 <ul class="normal">
 
                 <ul class="normal">
−
                     <li>Received sequencing results for  lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan) found frameshift mutation</li>
+
                     <li>Received sequencing results for  lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan) and found frameshift mutation</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
 
                         <li>8 other liquid cultures were miniprepped and screened and 2 successful results were sent off for sequencing</li>
 
                         <li>8 other liquid cultures were miniprepped and screened and 2 successful results were sent off for sequencing</li>
 
                     </ul>
 
                     </ul>
−
                     <li>Digestion and ligation of   lac-CTH1-ycf54-ChlM-trc-FNR-fdx + trc-POR-ChlP-DVR1-ChlG and backbone swap of trc-POR into Cam</li>
+
                     <li>Digestion and ligation of lac-CTH1-ycf54-ChlM-trc-FNR-fdx + trc-POR-ChlP-DVR1-ChlG and backbone swap of trc-POR into Cam</li>
−
                     <li>First round of transformations were unsuccessful for lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG, trc-POR (Cam) and trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 we deduced the issue was with the competent cells we used</li>
+
                     <li>First round of transformations were unsuccessful for lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG, trc-POR (Cam) and trc-ChlI1-ChlI2-ChlD-ChlH-GUN4; we deduced the issue was with the competent cells we used</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
−
                         <li>Commercial competent cells were used to retransform samples and succeeded</li>
+
                         <li>Commercial competent cells were used to re-transform samples and succeeded</li>
−
                         <li>8 Colonies from each plate were liquid cultured</li>
+
                         <li>8 Colonies from each plate were liquid-cultured</li>
 
                     </ul>
 
                     </ul>
 
                 </ul>
 
                 </ul>
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                     <li>Induced  trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 (Mg-Chelatase) plasmids into competent cells and used Fluorescence testing to check for enzyme expression and function (formation of Mg-PPIX which is fluorescent).</li>
 
                     <li>Induced  trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 (Mg-Chelatase) plasmids into competent cells and used Fluorescence testing to check for enzyme expression and function (formation of Mg-PPIX which is fluorescent).</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
−
                         <li>Growth was to too little to yield results currently.</li>
+
                         <li>Growth was too little to yield results at that moment.</li>
 
                     </ul>
 
                     </ul>
 
                     <li>We are performing Cas9 purification as part of our collaboration with NTU so we prepared all necessary solutions for Cas9 purification in preparation for the arrival of their biobricks</li>
 
                     <li>We are performing Cas9 purification as part of our collaboration with NTU so we prepared all necessary solutions for Cas9 purification in preparation for the arrival of their biobricks</li>
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                     <li>Performed the standard assembly of our last 2 parts together for our Chlorophyll plasmid:  trc-chlI1-ChlI2-D-trc-ChlH-GUN4 +  lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG</li>
 
                     <li>Performed the standard assembly of our last 2 parts together for our Chlorophyll plasmid:  trc-chlI1-ChlI2-D-trc-ChlH-GUN4 +  lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
−
                         <li>Screened by colony PCR which was able to narrow down our samples, we miniprepped hopefully samples and performed single double digests for screening</li>
+
                         <li>Screened by colony PCR which was able to narrow down our samples, we miniprepped samples and performed single/double digests for screening</li>
 
                     </ul>
 
                     </ul>
−
                     <li>Mg-PPIX induction testing showed high formation of Zn-PPIX which has a similar fluorescence to Mg-PPIX which dampens out any meaningful results</li>
+
                     <li>Mg-PPIX induction testing showed high formation of Zn-PPIX which had a similar fluorescence to Mg-PPIX. This dampened out any meaningful results</li>
 
                     <ul class="indented">
 
                     <ul class="indented">
−
                         <li>We have proceeded using M9 minimal salt plates for our induction experiments to reduce this problem</li>
+
                         <li>We proceeded using M9 minimal salt plates for our induction experiments to reduce this problem</li>
 
                     </ul>
 
                     </ul>
 
                     <li>Received constructs from NTU and transformed and liquid cultured them, then proceeded to begin with Cas 9 extraction</li>
 
                     <li>Received constructs from NTU and transformed and liquid cultured them, then proceeded to begin with Cas 9 extraction</li>

Revision as of 03:17, 11 October 2018


Wet lab notebook

  • Project planning - no wet lab work this week.
  • We went through lab safety induction to ensure safety procedures were followed at all times in the lab
  • We learnt how to perform digestion, ligation, transformation and how to make and run agarose gels
  • We used digestion to screen the Photosystem II (PSII) parts DCA, psbELJTB and psbMZtlwkOPQR. psbELJTB and psbMZtlwkOPQR showed successful digestion and correct sizes. DCA showed signs of contamination in all samples.
  • We decided to focus of the Chlorophyll plasmid rather than PS II. We proceeded with digestion and ligation of ChlD with ChlI2 as well as DRV1 with ChlG, both into Kanamycin backbones, following the 3A assembly protocol.
  • Renee, Areti and Karl also began the interlab study:
  • Made LB media + cam plates
  • Transformed Εscherichia coli DH5α cells with the interlab testing devices.Transformation was performed twice, due to poor colony growth in the LB + cam media at the first round of transformations.

Figure 1. Lorem ipsum.

  • We transformed the DVR1-ChlG and ChlD-ChlI2 ligation samples into competent DH5α E.coli cells. DVR1-ChlG had successful transformants which were then incubated in LB broth + Kan. ChlD-ChlI2 had given an unsuccessful transformation, so the process was repeated and was successful the second time. The samples were then liquid cultured.
  • Interlab experiments were completed this week:
  • Calibrations 1, 2 and 3 were completed early on in the week
  • Cell measurements (Abs600 and fluorescence) were taken as per the interlab protocol
  • Colony forming units per OD600 = 0.1 of the negative and positive control devices was also determined (in triplicates)
  • Set up backbone insertions for trc-POR, trc-ChlI1 (Digestion, ligation and transformation
  • Interlab experiments:
  • Colony forming units per OD600 = 0.1 of the negative and positive control devices was completed again.
  • Data analysis and submission.
  • The liquid cultures of DVR1-ChlG and ChlD-ChlI2 were miniprepped and digested (single, double digest) to screen for successful ligations by agarose gel electrophoresis (1% agarose).
  • The gel showed unsuccessful ligations so we switched our ligation technique to standard assembly and backbone swapping of necessary parts
  • We backbone-swapped DVR1 to Kan and were in the process of swapping ChlD, trc-POR and GUN4
  • Made up more plasmid stock of current parts by plasmid transformation, culturing and miniprep
  • Begun Standard assembly of DVR1 + ChlG
  • Miniprepped all samples
  • Begun induction and SDS-PAGE to compare lac-ChlH and trc-ChlH
  • Ran GUN4, ChlD and trc-POR backbone swaps as well as DVR1-ChlG on a 1% agarose gel
  • Sent off successful samples of each for sequencing
  • We inserted synthesised DNA biobricks into Amp and Cam backbones for trc-POR,YidC and trc-ChlI1, trc-ycf39-HliD
  • We then transformed, liquid-cultured, miniprepped and screened on a 1% agarose gel
  • We had successes for trc-ChlI1 in Amp, which we sent for sequencing confirmation
  • Proceeded with standard assembly for:
  • CTH1-ycf54-ChlM-ter + trc-FNR-fdx or trc-FNR
  • trc-ChlH + GUN4
  • trc-POR + ChlP
  • ChlI2 + ChlD

  • We sent CTH1-ycf54-ChlM-ter-trc-FNR-fdx as well as CTH1-ycf54-ChlM-ter-trc-FNR for sequencing
  • Sequencing came back for trc-ChlH to show that the promoter swap was unsuccessful. A reattempt of promoter swap was then undertaken
  • Transformed trc-POR-ChlP and ChlI2-ChlD
  • Screened trc-ChlI1 and sent 1 successful plasmid away for sequencing
  • Performed SDS-PAGE comparing trc-FNR-fdx and lac-FNR-fdx
  • Liquid-cultured, Miniprepped and screened trc-ChlH, trc-POR-ChlP, trc-ycf39-HliD, trc-FNR and trc-FNR-fdx and ChlI2-ChlD
    • sent ChlH, trc-POR, trc-ycf39-HliD, trc-FNR-fdx and ChlI2-ChlD plasmids for sequencing
  • Digestion/ligation for trc-POR+ChlP, ChlI1+ChlI2-ChlD, trc-ChlH-GUN4
  • Glycerol stock made for ChlH
  • Transformed, Liquid-cultured and miniprepped trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP
  • Made glycerol stock for trc-POR and trc-ChlH
  • Digested and screened trc-ChlH-GUN4, trc-FNR and trc-FNR-fdx. Only trc-FNR-fdx and trc-ChlH-GUN4 had successes, which were sent off for sequencing
  • Induced trc-POR cells and ran the cell lysate on SDS-PAGE
  • Digested and screened trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP on a 1% agarose gel and sent successful samples for sequencing
  • Backbone-swapped lac-CTH1-ycf54-ChlM (Kan)
  • Performed standard assembly on trc-POR-ChlP-DVR1-ChlG and sent successful ones for sequencing
  • Induced new trc-POR samples and ran cell lysate on an SDS-PAGE
  • Grew barley for protochlorophyllide extraction
  • Made up more plasmid stock of trc-POR
  • Made up glycerol stock of ChlI1
  • Redesigned and ordered sequencing primers
  • Tried to insert G-block for yidC and trc-FNR into ampicillin backbone, no successes from screened samples
  • Digestion/ Ligation of trc-ChlI1-ChlI2-ChlD + ChlH-GUN4. Backbone swap of trc-POR (switched to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR
  • Received sequencing results for trc-POR-ChlP and sent off trc-POR-ChlP-DVR1-ChlG samples for sequencing
  • Digested, ligated, transformed and liquid-cultured lac-CTH1-ycf54-ChlM + trc-FNR-fdx
  • Transformed trc-ChlI1-ChlI2-ChlD + trc-ChlH-GUN4 (shortened to trc-IH), Backbone-swapped trc-POR (to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR
    • Liquid-cultured, miniprepped and performed single/double screening of digests on a 1% agarose gel
    • Sent 3 IH samples and 1 of both trc-POR (Cam) and lac-CTH1-ycf54-ChlM-trc-POR for sequencing
  • Made induction plates and induced trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4 (Mg-Chelatase) to see if we could get Mg-PPIX formation
  • Digested and ligated trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 + lac-CTH1-ycf54-ChlM-trc-POR (Streamlined operon)
  • Bradford assay of trc-POR samples to determine concentration of protein in the supernatant and pellet fractions of the cell lysate
    • POR is a membrane-associated protein, more (total) dissolved proteins in the supernatant
  • Extract protochlorophyllide from etiolated seedlings of barley
    • C18 Sep-Pak used for further purification
  • Received sequencing results for trc-ChlI1-ChlI2-ChlD-ChlH-GUN4, discovered trc-ChlI1 had not ligated initially but all other genes were present and sequence was confirmed
    • Stopped streamline operon and digested and ligated trc-ChlI1 to ChlI2-ChlD-ChlH-GUN4
  • Miniprepped 7 liquid cultures of lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan)
    • Performed single/double digests then ran on 1.5% agarose gel
    • Sent the 1 successful plasmid off for sequencing
    • Liquid cultured 8 more colonies of lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan)
  • Performed a functional assay of trc-POR induced cells using protochlorophyllide as substrate
    • Results were inconclusive and redone by Professor Robert Willows
  • Received sequencing results for lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan) and found frameshift mutation
    • 8 other liquid cultures were miniprepped and screened and 2 successful results were sent off for sequencing
  • Digestion and ligation of lac-CTH1-ycf54-ChlM-trc-FNR-fdx + trc-POR-ChlP-DVR1-ChlG and backbone swap of trc-POR into Cam
  • First round of transformations were unsuccessful for lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG, trc-POR (Cam) and trc-ChlI1-ChlI2-ChlD-ChlH-GUN4; we deduced the issue was with the competent cells we used
    • Commercial competent cells were used to re-transform samples and succeeded
    • 8 Colonies from each plate were liquid-cultured
  • Miniprepped and screened through PCR and agarose gel: lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG, trc-POR (Cam) and trc-ChlI1-ChlI2-ChlD-ChlH-GUN4
    • Sent 2 samples of trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 and 4 samples of lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG for sequencing
    • trc-POR backbone swap did not work so we redigested the original G-Block DNA for backbone insertion into Cam.
  • Induced trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 (Mg-Chelatase) plasmids into competent cells and used Fluorescence testing to check for enzyme expression and function (formation of Mg-PPIX which is fluorescent).
    • Growth was too little to yield results at that moment.
  • We are performing Cas9 purification as part of our collaboration with NTU so we prepared all necessary solutions for Cas9 purification in preparation for the arrival of their biobricks
  • Performed the standard assembly of our last 2 parts together for our Chlorophyll plasmid: trc-chlI1-ChlI2-D-trc-ChlH-GUN4 + lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG
    • Screened by colony PCR which was able to narrow down our samples, we miniprepped samples and performed single/double digests for screening
  • Mg-PPIX induction testing showed high formation of Zn-PPIX which had a similar fluorescence to Mg-PPIX. This dampened out any meaningful results
    • We proceeded using M9 minimal salt plates for our induction experiments to reduce this problem
  • Received constructs from NTU and transformed and liquid cultured them, then proceeded to begin with Cas 9 extraction