Difference between revisions of "Team:Macquarie Australia/Notebook1CBP"

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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/ProjectOverview"><img src="https://static.igem.org/mediawiki/2015/a/a0/MqAust_1Project_v06a-150dpi.png" width="220px" alt="Link to Project page"></a></figure>
 
<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/ProjectOverview"><img src="https://static.igem.org/mediawiki/2015/a/a0/MqAust_1Project_v06a-150dpi.png" width="220px" alt="Link to Project page"></a></figure>
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Experiments"><img src="https://static.igem.org/mediawiki/2015/9/91/MqAust_BubbleProject_2Experiments.png" width="110px" alt="Link to Experiments &amp; Protocols page"></a></figure>
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Notebook"><img src="https://static.igem.org/mediawiki/2015/9/98/MqAust_BubbleProject_5Notebook.png" width="110px" alt="Link to Notebook page"></a></figure>
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<figure class="specialInline"><img src="https://static.igem.org/mediawiki/2015/0/06/MqAust_BubbleNotebook_1CBP.png" width="80px" alt="Chlorophyll Biosynthesis Pathway page"></figure>
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Notebook2PSB"><img src="https://static.igem.org/mediawiki/2015/f/f8/MqAust_BubbleNotebook_3PSB.png" width="80px" alt="Link to Photosystem II page"></a></figure>
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<figure class="specialInline"><a href="https://2015.igem.org/Team:Macquarie_Australia/Notebook4July"><img src="https://static.igem.org/mediawiki/2015/1/1d/MqAust_BubbleNotebook_4July.png" width="80px" alt="Link to Winter break page"></a></figure>
 
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Revision as of 12:07, 14 September 2015

Notebook 1 CBP
Link to Project page
Link to Project Description page
Link to Experiments & Protocols page
Link to Results page
Link to Design page
Link to Notebook page
Link to Safety page
Chlorophyll Biosynthesis Pathway page
Link to ChlH Gene page
Link to Photosystem II page
Link to Winter break page

Notebook - Chlorophyll Biosynthesis Pathway

This notebook includes the lab-work done to insert the genes to allow E. coli to transform protoporphyrin-IX to chlorophyll-a.

Thursday 30 July
  • Plasmid prep of:
    • Chlm + lac + LB-CAM (x4);
    • Chli1 + lac + CAM (x4);
    • lac + Chlp AMP (x3);
    • lac + YCF54 + AMP (x3);
    • lac + CTH1 + AMP (x3);
    • Plasto + lac + CAM (x4);
    • ChlD + lac + CAM (x4),
    • DVR1 + lac + CAM (x4);
    • lac + POK + AMP (x3)
    • lac+CHL1+YCF54 CAM - DNA Extraction
  • 4 Colonies - plasmid prep
  • Digested
    • 1 Enzyme - Linearised - EcoR1
    • 2 Enzymes - Double Digest - EcoR1 +Xba1
  • Next week: Run on gel
  • Plasmid prep of liquid cultures of lac composite parts (ChlD, ChlM, DVR1, Chli1, Plasto), followed by nanodrop.
  • Attempted to build lac composite parts
    • Digest 2ug of lac plasmid with SpeI and PstI followed by PCR cleanup
    • Digest 100ng of ChlD, ChlM, DVR1, Chli1 and Plasto with Xba1 and PstI using fast AP
    • Ligated 50ng of digested lac plasmid with gene parts in a gene:lac 3:1 molar ratio

Tuesday 4 August
  • Transformed ligations from the 30th of July
  • Ran gel of lac + CTH1 + YCF54 restriction digests

Wednesday 5 August
  • Restriction digest of plasmid preps from 30th July

Thursday 6 August
  • Plasmid prep of liquid cultures of lac + gene parts transformed on the 4th August.
  • Ran gel of restriction digests of lac + gene parts (ChlM, ChlD, ChlP, YCF54, CTH1, Plasto, Chli1, DVR1, POR, CTH1+YCF54) from yesterday.
  • Gel results made no sense so re-digested the above parts for a second round of screening and conducted a PCR using gene specific primers for all except YCF54 and Plasto.
  • Ran gel of second restriction digests and PCR products.
  • DNA Extraction and plasmid prep
    • ChlM CAM (4 colonies)
    • DVR1 CAM (5 colonies)
    • Plasto CAM (4 colonies)
  • Digested all with:
    • 1 Enzyme - Linearised - EcoR1
    • 2 Enzymes - Double Digest - EcoR1 +Pst1
  • Plasmid prep of: Chlm 1.1-1.6 (1.4 missing),Gun4+lac(2.1-2.3), and Gun4+lac (Amp)(3.1-3.2);
  • Digestion enzymes: Plasmids were digested using:
    • EcoRI
    • EcoRI + SpeI (Double digestion)
  • Samples were purified using gel purification.
  • Plasmid prep, and nanodrop, of:
    • Chld 1.1-1.4, ChlI1 2.1-2.4, ChlI1+lac 3.1-3.2, ChlI2+lac 4.1-4.2, Gun4+lac B1, Gun4+lac B2, Gun4+lac B1(sh), Gun4+lac B2 (sh)
  • Enzyme digestion followed by gel elecrophoresis
    • Single digest with EcoRI
    • Double digest with EcoRI + SpeI

Thursday 13 August
  • PCR amplification, followed by gel electrophoresis, to make composite parts of:
    • lac + ChlD
    • lac + Gun 4
    • lac + Chlm
    • lac + ChIg
  • BioBrick 3A Assembly method to make composite parts of:
    • lac + ChlD
    • lac + Gun4
    • lac + ChlM
    • plasto + ChlM
    • lac + ChlG
  • ~5ng of lac promoter plasmids were digested with SpeI and PstI and ~8ng were digested with EcoRI and PstI. These digested plasmids were then gel purified for use in the construction of the composite parts listed above.
  • Double Digest (XbaI + Pst1), followed by gel electrophoresis for purification, of:
    • ChlD
    • Gun4
    • ChlM x2
    • ChlG

Thursday 20 August
  • Digest of:
    • Chli2 (XbaI + Pst1)
    • Chli1 (SpeI + Pst1)
    • ChlD (SpeI + Pst1)
    • Plasto (SpeI + Pst1)
  • Plasmid preps of successful E. coli transformants from BioBrick 3A Assembly followed by PCR (due to low nucleic acid concentration) and gel electrophoresis of:
    • Lac + ChlM
    • Plasto + Chlm
  • Gel electrophoresis showed no products
  • Competent cells were transformed with Lac+ChlM composite gene parts and plasto+ChlM parts which were used in the original successful transformants
  • A number of ligations were undertaken in order to build a variety of composite parts. Firstly, we ligated 25ng of digested lac plasmid from last week with the following gene parts;
    • ChlD (75ng)
    • GUN4 (50ng)
    • ChlM (50ng)
    • ChlG (50ng)
  • These ligations occurred at 37°C for one hour, with an 80°C incubation to deactivate the enzyme. Once complete, the ligation products were transformed into competent cells and plated onto CAM media.
  • These digested parts listed above were also ligated as seen below:
    • 25ng of Chli1 + ChlD with 25ng of Chli2
    • 25ng of ChlM with 25ng of Plasto
    • 25ng of digested lac promoter from last week with 50ng of ChlG PCR product.
  • These ligation products were also transformed into competent cells and plated onto the appropriate media.