Team:Macquarie Australia/Notebook1CBP

Notebook 1 CBP

Chlorophyll Biosynthesis Pathway Notebook

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

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

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

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:

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)

~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:

Plasmid preps of successful E. coli transformants from BioBrick 3A Assembly followed by PCR (due to low nucleic acid concentration) and gel electrophoresis of:

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;

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:

These ligation products were also transformed into competent cells and plated onto the appropriate media.