Team:KU LEUVEN/Experiments

Experiments

Direct
manipulation Indirect
manipulation Primers

Direct manipulation: engineering of a cyanobacterium

Preparation of BG11 and S. elongatus UTEX 2973 growth conditions

Constructing a growth curve of S. elongatus UTEX 2973

Creating recombinant pAM2991 plasmids

Transformation of plasmids into E. coli using heatshock

Preparation and revival of glycerol stocks

Plasmid purification using miniprep

Indirect manipulation: engineering cyanophage S-TIP37

Preparation of ASW medium and growth of Synechococcus sp. WH8109

Phage propagation

Engineering of the S-TIP37 genome

Assembly verification

Utilized primers

Primers used for tail-PCR of signal peptides and sfGFP

	Forward Primer	Reverse Primer
Signal Peptide 1	caagcttgcatgcctgcaggtcgactctagagATGAACTTTCCGAGAGCGAGTC	CCGTGAACAGTTCTTCTCCTTTACTGGCTGTAGCCGCCGC
Signal Peptide 2	caagcttgcatgcctgcaggtcgactctagagATGAAGATTAAGACTGGCGCCCGGATC	CCGTGAACAGTTCTTCTCCTTTACTGGCAAGGGCCGACGCAC
Signal Peptide 3	caagcttgcatgcctgcaggtcgactctagagATGAAAAAGCGAGGCGCCTTTCTTG	CCGTGAACAGTTCTTCTCCTTTACTAGCAAAAACCGAAGCACAAGCG
sfGFP1	CGGCTACAGCCAGTAAAGGAGAAGAACTGTTCACGG	caatttcacacaggaaacagaccatggCTAGTGATGATGGTGGTGATGTTTGTACAGCTCATCCATGCCG
sfGFP2	GTGCGTCGGCCCTTGCCAGTAAAGGAGAAGAACTGTTCACGG	caatttcacacaggaaacagaccatggCTAGTGATGATGGTGGTGATGTTTGTACAGCTCATCCATGCCG
sfGFP3	CGCTTGTGCTTCGGTTTTTGCTAGTAAAGGAGAAGAACTGTTCACGG	caatttcacacaggaaacagaccatggCTAGTGATGATGGTGGTGATGTTTGTACAGCTCATCCATGCCG

Table 1: Primers used for Tail-PCR to create overhangs on sfGFP and signal peptides. Lowercase letters identify those basepairs corresponding to plasmid overhangs, while uppercase basepairs are lying on the insert itself.

Primers used for insert verification on the S-TIP37 genome

Location	Forward Primer	Reverse Primer
Lysis Cassette	gattacgatgactcaagcgac	cagaaatggtactacggcgag
Integrase	cagggacgacatcttgcaaac	cagaaatggtactacggcgag
Structural Cassette	ctacgccctgtgctacatc	gacattgaagcaacaccacc
After the Last Gene	gagcagtgtcgtacacagattg	gacccaccatatggctatggag

Table 2: Primers used for insert verification on the S-TIP37 genome, wrapping the integrated cassette. Both primers are situated on the S-TIP37 genome.