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

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.