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

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<h2>Chlorophyll Biosynthesis Pathway Notebook</h2>
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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>
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<h2>Notebook - Chlorophyll Biosynthesis Pathway</h2>
 
<p>This notebook includes the lab-work done to insert the genes to allow <i>E. coli</i> to transform protoporphyrin-IX to chlorophyll-<i>a</i>.</p>
 
<p>This notebook includes the lab-work done to insert the genes to allow <i>E. coli</i> to transform protoporphyrin-IX to chlorophyll-<i>a</i>.</p>
  

Revision as of 07:26, 10 September 2015

Notebook 1 CBP
Link to Project 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 Wet-lab
  • 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 13th 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.