Still looking for assay for FAEEs

How are we relevant?

Algae making biofuel has issues (contamination, economy). We could beat enzymatic hydrolysis because we consolidate the enzyme synthesis and product production in one. 

Biobricks!

Are very old and stuck in the past. We should use the biobrick base vector but modify it to be compatible with new cloning methods. 

Biobrick vector contains antibiotic resistance marker and an origin of replication, and a number of restriction enzyme sites. We could replace the resistance marker with a resistance gene and a specific ori. Make a biobrick vector where we swap in MoClo (Modular Cloning( sites. MoClo allows simultaneous rather than just sequential operations/digestions. 

TOOLS

1. restriction enzymes to cut
2. Ligase to stick together
3. Phosphatase --> phosphate groups
4. PCR: heat double stranded DNA at 94 degrees for 30 secs and the strands come apart. Unique flanking primers. We need the primers to have the same melting temperature, say 60 degrees (geneious can estimate this), then cool mixture to 58 degrees. Then use Taq polymerase to bind to the primers and march down the single stranded DNA adding the complementary nucleotides. (72 degrees- primers don't melt because even at 58 degrees Taq starts adding more nucleotides and thus the dissociation temperature increases.) Choosing extension time is important - Taq has a finite speed. Repeat until you have enough. Exponential increase until you run out of primers and nucleotides. Polymerases get denatured over time. 
5. Cas9 = targetable restriction enzyme. really easy to make new pieces of DNA. Can have 20-nucleotide recognition sites
6. Site-directed mutagenesis: you can get rid of recognition sites using silent mutations. Melt to separate into two circular single strands, then add primers which aren't direct matches to introduce the silent differences in nucleotides. 
7. IDT 
   1. -primers <60 BP. Bead-stick on nucleotides one at a time then drop it off the bead to produce your primer. single stranded and short
   2. gBlocks: double stranded and long up to 2.5kb overlapping DNA strands in a block.

Ligase links the groups together- 

Issues with old way: the complementary ends on the plasmid could stick to eachother. So use 2 different restriction enzymes so the restriction sites are different for each end so it can't bind itself. 

Use selection: each end of plasmid contains part of gene toxic to E Coli, so if they stick together E Coli dies. Select using multiple resistances. 

Screen using GFP. Add LacZalpha marker. It is a complex that turns galactose into lactose and glucose (or the other way round?) Put on plate with X-gal which turns blue when LacZ is floating around. The colonies which aren't blue are the ones we want- the LacZ should've been split apart due to the gene being inserted.