Plasmid backbones/Operation

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Reshma's low and medium copy plasmid backbones • Additional low and medium copy plasmid backbones Inducible copy number plasmid backbones • Multi-host plasmid backbones • Eukaryotic plasmid backbones

Plasmids are circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. Plasmid DNA is easily purified from cells, manipulated using common lab techniques and incorporated into cells. Most BioBrick parts in the Registry are maintained and propagated on plasmids. Thus, construction of BioBrick parts, devices and systems usually requires working with plasmids.

Note: In the Registry, plasmids are made up of two distinct components:

1. the BioBrick part, device or system that is located in the BioBrick cloning site, between (and excluding) the BioBrick prefix and suffix.
2. the plasmid backbone which propagates the BioBrick part. The plasmid backbone is defined as the sequence beginning with the BioBrick suffix, including the replication origin and antibiotic resistance marker, and ending with the BioBrick prefix. [Note that the plasmid backbone itself can be composed of BioBrick parts.]

Many BioBrick parts in the Registry are maintained on more than one plasmid backbone!

Once you have assembled your BioBrick device or system, you're then ready to "run" it in your chassis of choice. You might be interested in characterizing its performance, demo'ing your engineered systems to your friends, or documenting its performance for the iGEM jamboree or a publication.

Regardless, it is oftentimes helpful to operate your system on a low or medium copy plasmid backbone. By lowering the DNA copy number of your BioBrick device or system, it will consume less of the cell's resources and thus you can operate more devices in parallel and potentially achieve more reliable system performance.

Note, however, that low or medium copy plasmid backbones tend to have reduced resource consumption because fewer transcripts and proteins are made. Thus, characterization of device or system performance can be more difficult, since you are measuring a smaller number of molecules.

Reshma's low and medium copy operation plasmid backbones

All of these "operation" plasmid backbones have a common set of features.

1. A complete BioBrick® cloning site for easy cloning and assembly of BioBrick® parts.
2. A low or medium copy replication origin to reduce consumption of cellular resources during device or system operation.
3. Forward and reverse terminators both upstream and downstream of the BioBrick® cloning site to insulate the vector from read-through transcription originating in the cloned BioBrick® device or system and to insulate the cloned BioBrick® system from the vector.
4. Stop codons in all reading frames to prevent inadvertent translation into or out of the BioBrick cloning site.
5. Primer binding sites for the standard BioBrick® verification primers VF2 (BBa_G00100) and VR (BBa_G00101). These primers are located for convenient sequencing and [http://openwetware.org/wiki/Colony_PCR screening by colony PCR] of cloned BioBrick® devices and systems.

Plasmid backbones are distributed by the Registry with a default insert. There are just a handful of default plasmid inserts used in the Registry. All of the available plasmid backbones include the RFP expression cassette (BBa_J04450) as the default plasmid insert within the BioBrick® cloning site. The RFP cassette enables red/white screening of colonies and ensures that when assembling two BioBrick® parts together, the uncut plasmid (or a plasmid that get the RFP cassette as insert) will be red on the plate whereas the correct colonies will be white.

Plasmid backbones with the plasmid insert of BBa_I52002 also include a high copy replication origin in the default insert. Thus, these plasmid backbones are easily [http://openwetware.org/wiki/Miniprep/Qiagen_kit purified in large quantities]. Cloning or assembly of a BioBrick® part into the BioBrick® cloning site of the plasmid backbone eliminates the default insert returning the plasmid to the control of the replication origin in the plasmid backbone.

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Name	Description	Resistance	Replicon	Copy number	Chassis	Length
BBa_K1362095	pSB4A50: Low copy BioBrick cloning/expression backbone carrying Amp resistance	A	pSC101	~5		3446
BBa_K1362096	pSB4C50: Low(?) copy BioBrick cloning/expression backbone carrying Cm resistance	C	pSC101	unsure		3408
BBa_K1362097	Low copy BioBrick expression backbone carrying Kan resistance	K	pSC101	~5		3470
BBa_K864000	Low copy BioBrick standard vector	A	pSC101	~5		3639
BBa_K864001	Low copy BioBrick standard vector	C	pSC101	~5		3466
BBa_K864002	Low copy BioBrick standard vector	K	pSC101	~5		3663
BBa_K864003	Low copy BioBrick standard vector	S	pSC101	~5		3793
BBa_K864004	Low copy BioBrick standard vector	A	pSC101	~5		3708
BBa_K864005	Low copy BioBrick standard vector	C	pSC101	~5		3534
BBa_K864006	Low copy BioBrick standard vector	K	pSC101	~5		3732
BBa_K864007	Low copy BioBrick standard vector	S	pSC101	~5		3861
BBa_K864008	Low copy BioBrick standard vector with LacI	A	pSC101	~5		4819
BBa_K864009	Low copy BioBrick standard vector with LacI	C	pSC101	~5		4646
BBa_K864010	Low copy BioBrick standard vector with LacI	K	pSC101	~5		4843
BBa_K864011	Low copy BioBrick standard vector with LacI	S	pSC101	~5		4973
BBa_K864012	Low copy BioBrick standard vector with LacI	A	pSC101	~5		4888
BBa_K864013	Low copy BioBrick standard vector with LacI	C	pSC101	~5		4714
BBa_K864014	Low copy BioBrick standard vector with LacI	K	pSC101	~5		4912
BBa_K864015	Low copy BioBrick standard vector with LacI	S	pSC101	~5		5041
BBa_K864016	Low copy BioBrick temperature sensitive standard vector	A	pSC101ts	~5		3639
BBa_K864017	Low copy BioBrick temperature sensitive standard vector	C	pSC101ts	~5		3466
BBa_K864018	Low copy BioBrick temperature sensitive standard vector	K	pSC101ts	~5		3663
BBa_K864019	Low copy BioBrick temperature sensitive standard vector	S	pSC101ts	~5		3793
BBa_K864020	Low copy BioBrick temperature sensitive standard vector	A	pSC101ts	~5		3708
BBa_K864021	Low copy BioBrick temperature sensitive standard vector	C	pSC101ts	~5		3534
BBa_K864022	Low copy BioBrick temperature sensitive standard vector	K	pSC101ts	~5		3732
BBa_K864023	Low copy BioBrick temperature sensitive standard vector	S	pSC101ts	~5		3861
pSB3C5	Low to medium copy BioBrick standard vector	C	p15A	10-12		2738
pSB3K5	Low to medium copy BioBrick standard vector	K	p15A	10-12		2936
pSB3T5	Low to medium copy BioBrick standard vector	T	p15A	10-12		3252
pSB4A5	Low copy BioBrick standard vector	A	pSC101	~5		3395
pSB4C5	Low copy BioBrick standard vector	C	pSC101	~5		3221
pSB4K5	Low copy BioBrick standard vector	K	pSC101	~5		3419
pSB4T5	Low copy BioBrick standard vector	T	pSC101	~5		3735

Reshma Shetty, Drew Endy, and Tom Knight constructed this set of plasmid backbones. You can read more about the design and construction of these plasmid backbones in their open-access paper, [http://www.jbioleng.org/content/2/1/5 Engineering BioBrick vectors from BioBrick parts] published in the Journal of Biological Engineering.

Additional low and medium copy operation plasmid backbones

There are additional low and medium copy BioBrick® plasmid backbones available, all of which have a complete BioBrick cloning site.

	Stefan Luzi constructed the three plasmid backbones (pSB6A1, pSB4C1, and pSB3K1) as a member of the [http://2007.igem.org/ETHZ 2007 ETH Zurich iGEM team].
	Jeff Tabor constructed plasmid backbone BBa_J64100 in Chris Voigt's lab.
	Reshma Shetty constructed plasmid backbone pSB3K3 in Tom Knight's lab.

More...

Name	Description	Resistance	Replicon	Copy number	Chassis	Length
BBa_J61031	Amplifiable BAC plasmid	K	ColE1/R6K/F			10662
BBa_K125000	Broad host range BioBrick plasmid derived from pRL1383a with low to medium copy number					9047
BBa_K592200	Low to medium copy BioBrick standard vector	S	p15A	10-12		3137
pSB3K1	pACYC177 converted into BioBrick vector (formerly BBa_I739202)	K	p15A	10-12		3064
pSB3K3	Low to medium copy BioBrick standard vector	K	p15A	10-12		2750
pSB4C1	pCK01 converted into BioBrick vector (formerly BBa_I739202)	C	pSC101	5-12		3155
pSB6A1	pBR322 converted to BioBrick vector (formally BBa_I739201)	A	pMB1	15-20		4022

Inducible copy number operation plasmid backbones

Sometimes it is nice to have the best of both worlds. For example, you'd usually like a BioBrick® part to propagate at low copy, but you'd also like to switch the part over to high copy when you're interested in purifying plasmid DNA. The solution is an inducible copy number plasmid backbone. Both plasmid backbones listed below have a high copy replication origin under the control of a LacI-repressible promoter. When the plasmid is propagated in an E. coli strain that expresses the repressor lacI in high quantities, such as D1210 (or BBa_V1003), the plasmid is maintained at low copy by the F' origin. To increase the plasmid copy number, simply add the small molecule [http://openwetware.org/wiki/IPTG IPTG] to induce the LacI-repressible promoter controlling the high copy number origin.

• Note: because these inducible copy number vectors rely on LacI for control of the high copy number origin, they are incompatible with any BioBrick® part, device, or system that include lacI or a LacI-regulatable promoter.

The plasmid backbones listed below have a common set of features.

1. A complete BioBrick® cloning site for easy cloning and assembly of BioBrick parts.
2. Terminators flanking the BioBrick® cloning site to insulate the vector from read-through transcription originating in the cloned BioBrick® part, device or system.
3. Primer binding sites for the standard BioBrick® verification primers VF2 (BBa_G00100) and VR (BBa_G00101). These primers are located for convenient sequencing and [http://openwetware.org/wiki/Colony_PCR screening by colony PCR] of cloned BioBrick® parts, devices, and systems.

Plasmid backbones are distributed by the Registry with a default insert. There are just a handful of default plasmid inserts used in the Registry. Many the available plasmid backbones have the ccdB positive selection marker (BBa_P1010) as the default plasmid insert within the BioBrick® cloning site. The ccdB gene ensures that when assembling two BioBrick® parts together, the uncut plasmid is not transformed. However, inclusion of the ccdB gene means that these vectors must be propagated in a ccdB tolerant strain, such as E. coli strain DB3.1 (BBa_V1005).

More...

Name	Description	Resistance	Replicon	Copy number	Chassis	Length
pSB2K3	Inducible copy number BioBrick plasmid	K	F' and P1 lytic	<10 or >100	lacIq like BBa_V1003	4425
pSB2K4	pSB2K3-derived BioBrick plasmid free of many restriction sites	K	F' and P1 lytic	<10 or >100	lacIq like BBa_V1003	4467

Sri Kosuri and Leon Chan constructed plasmid backbone pSB2K3 in Drew Endy's lab based on the [http://genome.bnl.gov/Vectors/pscans.php pSCANS] vector from Brookhaven National Lab. They, together with Austin Che, constructed pSB2K4.

Multi-host operation plasmid backbones

Sarah Hollingshead constructed this multi-host BioBrick plasmid backbone as a member of the [http://2007.igem.org/Edinburgh 2007 University of Edinburgh iGEM team] from the pTG262 vector generously provided by Dr Mike Gasson & Dr. Claire Shearman of the Institute of Food Research in Norwich. The Edinburgh team used the plasmid backbone as a shuttle plasmid backbone between lactic acid bacteria and E. coli. It has a gram positive replication origin.

There are no parts for this table

Eukaryotic operation plasmid backbones

Monika Ciglic constructed this E. coli high copy plasmid backbone with a eukaryotic terminator as a member of the [http://2006.igem.org/Ljubljana,_Slovenia_2006 2006 Slovenija iGEM team] from pSB1AK3.

There are no parts for this table

Notes

• Part:BBa_I742123 - physical location unknown?

References

<biblio>

1. Hershfield74 pmid=4610576
2. Mandecki90 pmid=2227445
3. Viera82 pmid=6295879
4. YanishPerron85 pmid=2985470
5. Messing91 pmid=2055478
6. Chang74 pmid=4598290
7. Cohen77 pmid=334752
8. Cabello76 pmid=765836
9. Sugiura93 pmid=8376344
10. Miller95 pmid=7665462
11. Bolivar77 pmid=344136
12. Bolibar77b pmid=344137
13. Chang78 pmid=149110
14. Murakami87 pmid=3031019
15. Uga89 pmid=10393200
16. Kawasaki96 pmid=9003285
17. Stueber82 pmid=6327267
18. Bernard84 pmid=7926841

</biblio>