Methods

Inserting the BBa_K977000 pyruvate kinase gene into BBa_K125000 vector required a number of different steps and techniques.

Because the vector already contained a usable promoter, the main procedure involved four different methods.

• Using site-directed mutagenesis to remove a naturally-occurring PstI restriction site in the pyruvate kinase gene
• Using PCR to “flank” the pyruvate kinase gene with the BioBrick Standard Restiction Enzyme sites
• Using 3A Assembly to insert the new part into the BBa_K125000 vector
• Transforming the cyanobacteria

PCR AND SITE-DIRECTED MUTAGENSIS
Site-directed mutagenesis is a method for altering the sequence of DNA and can be used to produce both point and frame-shift mutations. In this case, it was possible to take advantage of codon redundancy and alter the DNA sequence without altering the amino acid sequence. It was necessary to do site-direct mutagenesis because the pyruvate kinase gene contains in its sequence two PstI restriction sites.
Because PSTI is part of biobrick assembly, it was necessary to use site-directed mutagenesis to remove these sites prior to cleaving and ligating the part. Otherwise PstI would chop the gene to pieces whenever we (or anybody else) tried to assemble anything with it.
Primer design was very simple, as the primers were identical to the gene except that they contained the desired mutations. Again, the primers were about 20 bp to ensure that they would only attach to one region along the gene.
Forward primer
Template strand- 5’- ctcatctccctgcaggtgaagcag 3’
Primer -5’- ctcatctccctccaggtgaagcag 3’
(Restriction sites are in red, point mutation is in blue)
Reverse primer
Again, the reverse primer necessitated the use of the reverse compliment of the original sequence.
Template strand 5’- CTGCTTCACCTGCAGGGAGATGAG -3’
Primer 5’- CTGCTTCACCTGCAGGGAGATGAG 3’

PCR In order to get the pyruvate kinase gene to conform to Biobrick Standards, it was necessary to add the four Biobrick restriction sites.
This was done with a variation on PCR annealing and extension per the instructions given by the Registry. Theoretically, this method was simple. All it required was forwards and reverse primers that consisted of the BioBrick prefixes and suffixes and the first twenty base pairs of the gene.
Forward primer
5’- GTTTCTTGGAATTCGCGGCCGCTTCTAGATG TCAAAGTCCCACAGTGAAGC -3’
Reverse Primer
To orient the sequence 5’-to-3’, it was necessary to use the reverse compliment of the last twenty base pairs of the gene.
5’- GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTACGGGACGGGCACCACGCGCA -3’
(Red is the tails, green is the gene)
After this, it would be possible to proceed with PCR as normal, and though the region of restriction sites would “hang off” in the first round of PCR, they would be added to the gene as “flanks” in subsequent rounds as the extension stage would transcribe the flanks on as well as the gene’s DNA. However, a number of challenges presented themselves.
At 51 and 55 bp, the length of the primers far exceeded the typical 40-45 bp. The long “free ends” that contained the restriction sites threatened to rip the section of matching sequence away from the template strand. It proved impossible to shorten these primers while still including the standard restriction site. Nonetheless, the primers were ordered and the experiment was attempted.
In the event of the failure of these super-long primers to anneal properly, it would also be possible to construct the restriction site flanks through multiple rounds of PCR, though this would be more time-consuming.

GIBSON ASSEMBLY
TIt would have also been possible to construct the PCL-5 gene entirely from oligos using Gibson assembly. The artificial synthesis of the approximately 1500-bp gene would require four oligos of single-stranded DNA. The ends of these oligos would contain small section of overlapping sequence, and could be annealed together, with DNA polymerase and DNA ligase completing and sealing the double strand. After this point, it would be possible to proceed with standard PCR.
The primary advantages of Gibson assembly included the fact that it would be possible to build the BioBrick restriction sites and altered PstI sites into the oligos. This is far simpler than using site-directed mutagenesis and PCR; however, ordering (and waiting for the synthesis of) such long oligos would actually be more time-consuming than using PCR and site-directed mutagenesis.
TINKER CELL A program called TinkerCell was utilized to help visualize the steps taken through glycolysis and the fatty-acid pathway to create alkanes. The image created in TinkerCell allows one to see each step in the modification of a molecule. Beginning with glucose and ending with alkanes, the image shows what the molecule becomes, which enzyme catalyzes the reaction, what type of reaction occurs, what is required for the reaction to occur, and the other products of the reaction.