Basic Molecular Biology Techniques:

PCR using Q5 Hi-Fi DNA polymerase Master Mix

Primer 1: 5’-GCACTCACCATGGGTACTGCAGAATTCGCGGCCGCTTCT-3’

Primer 2: 5’-TAGTGGTACCGCATGCCTGCACTGCAGCGGCCGCTACT-3’

Reaction setup:

The template DNA should keep an amount of 1-1000 ng

Thermocycler conditions

Clarification:

1. The sequences in red are where the primers anneal with the biobrick affixes. And the 5’overhangs side with the sequences in red are designed for Gibson Assembly with linearized pNZ8148 digested by PstI due to a 21-nucleotide overlap between them.
2. The reason of setting two different cycles is that, at the beginning of PCR, the annealed parts between primers and templates are only sequences in red. As PCR proceeds, 5’overhangs are added to the entire genes, so the rest reaction of PCR becomes the one between the whole primers and the whole genes, because of which the Tm varies compared to the former.

Applications in this project: Amplification of BBa_K2309028, BBa_K2309003 and BBa_K2309004

PCR purification using QIAquick PCR purification kit

1. Add 500 μl Qiagen buffer PB
2. Spin through a column twice, discard flow-through
3. Wash 1x with 700 μl buffer PB
4. Wash 2x with 760 μl buffer PE
5. Discard liquid, spin dry at 17000g for 3 min
6. Elute into a new tube twice with 50 μl of TE (100 μl total)

(Can be also referred to from iGEM protocol)

Gel extraction

Reagents:

1.5 ml micro-centrifuge tube; 2 ml collection tube; a QIAquick spin column; Isopropanol (100%); Buffer QG; Buffer PE; Buffer EB

Procedure:

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 μl, load and spin/apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow through and place the QIAquick column back into the same tube.

Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt- ended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.

8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.

Gibson Assembly

Digestion (NEB enzymes)

Ligation

Transformation of plasmids in E.coli DH5ɑ

Isolation of plasmid in E.coli DH5ɑ

Preparation of competent Lactococcus lactis

Electroporation for L. lactis

Plasmid DNA isolation from Lactococcus lactis

Anti-microbial peptides assay:

Nisin induction of gene expression in Lactococcus lactis: