Difference between revisions of "Part:BBa K1033207"

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−	[[File:Uppsala2013 chromo.JPG|900px|<i>E. coli</i> with pSBLbC containing RFP]] [[File:IMG 6644.JPG|900px|<i>Lactobacillus reuteri</i> 100-23 transformed with pSBLbC]]	+	[[File:Uppsala2013 chromo.JPG]]
	<i><b>Fig 1:</b> E. coli D5-alpha carrying pSBLbC with blue chromoprotein [[Part:BBa_K1033282|amilCP, and lactobacillus promotor CP29.]]</i>		<i><b>Fig 1:</b> E. coli D5-alpha carrying pSBLbC with blue chromoprotein [[Part:BBa_K1033282|amilCP, and lactobacillus promotor CP29.]]</i>

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Revision as of 00:14, 4 October 2013

Shuttle vector pSBLbE for E. coli and Lactobacillus

The backbone pSBLbC is a shuttle vector between E. coli, Lacotbacillus, and probably other lactic acid bacteria (LAB) like Lactococcus lactis. It has been used for subcloning in E. coli and to express a the chromoprotein amilCP.

Usage and Biology

Its replicon is known from litterature 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 very useful to do preliminary work in E. coli, and transfer finished constructs toLAB. But for that use we recommend the version with erythromycin resistance (pSBLbE) since that has been successfully used to transform Lactobacillus as well.

File:Uppsala2013 chromo.JPG

Fig 1: E. coli D5-alpha carrying pSBLbC with blue chromoprotein amilCP, and lactobacillus promotor CP29.

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 also changed the promotor of the chloramphenicol resistance cassette to one that would initiate transcription effectively in Lactobacillus. We tried several constitutive promotors but finally got CP29 to work.

See design subpage for more details.

Results

We have successfully subcloned small constructs into pSBLbC and used it to transform E. coli D5-alpha. Judging from levels of expression, copy number in E. coli is lower than pSB3K3. Despite several attempts we have not managed to transform Lactobacillus reuteri or Lactobacillus plantarum. We strongly suspect the resistance cassette is at fault since positive controls on antibiotic free agar plates have grown, but we have not had enough time to work out a solution. Please see our shuttle vector with erythromycin resistance, which has met with more success in that area.

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