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Applications of BBa_K341456

Expression of Kanamycin resistance gene

The kanamycin resistance gene is cloned from plasmid pKD13, under Tn5 Promoter, which renders constitutive kanamycin resistance gene expression.

E. Coli transformed with this part plasmid can grow and be selected with LB media with Kanamycin, thus proving the expression of kanamycin resistance gene.

Recombination machinery

This part is an Insertion Fragment of shuffling repertoire in the in vivo Recombination System of Tsinghua iGEM Team 2010.

The kanamycin resistance gene is flanked by a pair of landing pad, which designates the specific site for integration into E. Coli chromosome during homologous recombination. For the recombination events to occur, double strand break is first generated with I-SceI endonuclease, which cuts specifically at both ends of the segment.

Recombination protocol

In order for this part to be workable as a recombination shuffler, a modified E. Coli strain must be used. Besides, the helper plasmid harboring the I-Scel endonuclease and the RecA recombinase must be introduced to the E. Coli as well.

The E. Coli strain contains the corresponding landing pads flanking the kanamycin resistance gene, introduced by lambda att recombination.

After co-transformation with helper plasmid, the strain is cultured in LB media with L-arabinose. The E. Coli is then diluted 1e4 and plated onto media containing IPTG and one of the antibiotics, Kanamycin, tetracyclin or cholraphenicol.

Recombination Confirmation

Colonies grew on plates with kanamycin after our recombination induction.

Plated on media with kanamycine

Plated on media with tetracyclin

Later, we used the sequence flanking the insertion site of the E. Coli chromosome in addition to the specific sequence in kanamycin resistance gene as primers for PCR amplification. Thus, it will verify the insertion of the kanamycin resistance gene into the E. Coli chromosome after recombination.

PCR checking：Upper primer is in the E.coli genome, 500bp before att site, while the downer primer is in the landing pad. The positive result comes out only when the recombination occurs, shown as the figure. The left six lane are belong to a group of Negative Control.

Kanamycin resistance gene is 700bp and thus the colonies on the kanamycin plates have all incoporated kanamycin resistance gene into the corresponding site of E. Coli chormosome, which indicates that the design works.

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