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From 2012.igem.org

week 12: 16 july - 20 july

Office work

Lab work

Biobrick BBa_J04450 was cloned and miniprepped.

Bricking Hepatitis B

Tuesday:

• Digestion:

of the Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1 of BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1 of BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1

• PCR purification on the digest as a replacement for the heat inactivation

Wednesday:

• Ligation:

of the Hepatitis B PCR product with BBa_J04500 of the Hepatitis B product with BBa_PSB1K3.ml The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)

• Electrotransformation in E.coli
• Grow transformed E.coli on plates containing kanamycin

A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)

Thursday:

• Colony PCR

of 20 colonies transformed with the Hep B + BBa_J04500 construct of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct

Friday:

• gel electrophoresis (1% agarose gel) of the colony PCR samples

colony PCR of 20 colonies transformed with the Hep B + BBa_J04500:

the colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts

TuYV

• Mach1 electrocompetent E. coli cells were transformed with last week's ligation mixtures.

• 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 E. coli cells. After two attempts and a colony PCR, results were still unsatisfying.

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