Difference between revisions of "Part:BBa K1033207"

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	<i><b>Fig. 1</b> E. coli D5-alpha expressing RFP in our plasmid pSBLbE on 250 µg/ml erythromycin LB-agar plates.</i>		<i><b>Fig. 1</b> E. coli D5-alpha expressing RFP in our plasmid pSBLbE on 250 µg/ml erythromycin LB-agar plates.</i>
−	<i><Fig. 2</b> L. reuteri 100-23 with (left) and without (right) pSBLbE on 5 µg/ml erythromycin MRS-plates.</i>	+	<i><b><Fig. 2</b> L. reuteri 100-23 with (left) and without (right) pSBLbE on 5 µg/ml erythromycin MRS-plates.</i>
	=== References ===		=== References ===

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Revision as of 12:00, 4 October 2013

Shuttle vector pSBLbE for E. coli and Lactobacillus

The backbone pSBLbE is a shuttle vector between E. coli, Lacotbacillus. It has been grown in E. coli and used to transform Lactobacillus reuteri. It appears to be a low copy plasmid in E. coli, with a higher copy number in Lactobacillus, which is according to expectations.

Usage and Biology

The replicon used is known from literature to replicate in a wide range of gram positive and gram negative species^[1], and the backbone is meant to be used for work in E. coli and different LAB when it is necessary to do preliminary work in E. coli, and transfer finished constructs to LAB.

Construction

It was made by replacing the replicon of the BioBrick compatible plasmid pSB4C15 with a broad range replicon from the engineered plasmid pJP059. The replicon is also referred to as pSH71 and is related to pWV01 and the same family of rolling circle replicating plasmids.^[2]

We have changed the resistance cassette to one conferring erythromycin resistance, taken from the reuteri plasmid [http://www.ncbi.nlm.nih.gov/nuccore/AY556392.1 pLUL631]. It is easy to change resistance again thanks to the extra cloning sites of pSB4C15.

Results

The vector has been verified to work in E. coli and to provide resistance against 250 µg/ml erythromycin on LB-agar plates (though the erythromycin we have used is old, and a lower concentration than that may be appropriate for general use). The copy number has been estimated to be relatively low, judging by initially low expression of RFP.

It has also been shown to grow in Lactobacillus reuteri 100-23 on MRS-plates containing 5 µg/ml erythromycin, giving resistance to the transformed cells while negative controls were unable to surive. Here the copy number has been hard to assess, since the RFP currently used is not optimal for Lactobacillus.

Fig. 1 E. coli D5-alpha expressing RFP in our plasmid pSBLbE on 250 µg/ml erythromycin LB-agar plates.

<Fig. 2 L. reuteri 100-23 with (left) and without (right) pSBLbE on 5 µg/ml erythromycin MRS-plates.

References

^[1] I. Pérez-Arellano, M. Zúñiga, and G. Pérez-Martínez (2001), [http://www.sciencedirect.com/science/article/pii/S0147619X01915318 Construction of Compatible Wide-Host-Range Shuttle Vectors for Lactic Acid Bacteria and Escherichia coli], Plasmid 46 (2) 106-116. ^[2] I. Pérez-Arellano, M. Zúñiga, and G. Pérez-Martínez (2001), [http://www.sciencedirect.com/science/article/pii/S0147619X01915318 Construction of Compatible Wide-Host-Range Shuttle Vectors for Lactic Acid Bacteria and Escherichia coli], Plasmid 46 (2) 106-116.

Sequence and Features