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pMeca Plasmid
Wayne A. Parrott, PhD – 7/9/15
University of Georgia
What is pMECA?
pMECA is a cloning vector which contains 44 unique restriction sites, including 9 rare-cutters, all within
the 230-bp polylinker. Traditional blue/white screening can be used to identify bacterial colonies with
inserts cloned into the vector. Alternatively, bacterial colonies whose vectors contain inserts grow much
faster than those without, permitting the size of the bacterial colonies to be used to determine those
which contain inserts.
Strains that have been transformed with the vector:
DH5alpha, JM109, HB101, Top10 (Invitrogen XL-1 blue (Stratagene), DH5alpha (MCR)
Potential limitations:
Strain DH10B has been reported to not be transformable with pMECA. That appears to not be the case
once pMECA has an insert. This can be an advantage in that only pMECA clones containing an insert
grow well in this strain. We recommend transforming the vector into DH5alpha to grow up stock
plasmid. Then this stock can be used to clone into and transformed into DH10B. In theory only the
clones that appear on the plate should be recombinant.
The observation that XbaI digests pMECA poorly (if at all) can be explained by overlapping Dam
methylation. The site is preceded by GA to give the sequence 5'-GATCTAGA-3' (XbaI site underlined). The
complementary strand is 5'-TCTAGA*TC-3' (methylated A residue is marked with an asterisk,
methylation sequence is in bold). NEB recommends growing the plasmid in strain GM2163 (catalog #
401-P) a Dam minus strain. They claim that it is an OK strain to use for plasmid preps even though it is
endA1 positive.
Small colony size & slow growth:
The phenotype expected from this vector is slow growth (small colonies). This growth characteristic is
both on solid and in liquid media. Expect the cultures to take 24-48 hrs to appear on plates or to reach
log phase in culture. However, this is the advantage of the vector. When the MCS is disrupted the
growth of the cultures is normal (like that of pUC19).
Variable colony size-- which to use:
"We have transformed this plasmid [without an insert] into DH5 alpha and found two different sizes of
colonies. The majority of them are very very small and only a few of them are larger. Which colony (very
small one vs the bigger one) we should for the cloning experiments?"
Tip: Select the small colonies that appear on the plate and grow them up to make stocks. However, once
the plasmid has an insert, select the large colonies.
Pointers:
We would suggest for those first using the vector to plate the transformed cells on LB/Amp with IPTG
and X-gal. Once you become familar with the growth characteristics, then remove the use of IPTG and Xgal. Bacteria transformed with the vector will also take longer to appear blue. One trick is to place the
plates in the refrigerator (after growth at 37C) for several hours to better see the blue phenotype. Again,
once you see how easy it is to pick with size, you won't need to wait a long time for the small colonies to
grow or become blue, just select the large colonies on the plate after 16 hrs of growth. We did not ever
need to use chloramphenicol for the production of plasmid, but some may find it necessary.
Where can I find the sequence for the pMECA multiple cloning site?
GenBank ID: AF017063
What are the references for pMECA?
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
Thomson, J.M., and W.A. Parrott. 1998. pMECA: A size-based, blue/white selection multiple
common and rare-cutter general cloning and transcription vector. BioTechniques. 24:922-927
Parrott, WA. "Transformation Vector System." US Patent Number 6,096,523; Aug 1, 2000.
Pending applications in several jurisdictions – University of Georgia Research Foundation, Inc.
What are some references illustrating the use and potential of pMECA?
1.Hartwich H, Nothwang HG. An easy and versatile 2-step protocol for targeted modification and
subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids. BMC Res
Notes. 2012 Mar 20;5:156. doi: 10.1186/1756-0500-5-156
2.Huang YH, Cozzitorto JA, Richards NG, Eltoukhy AA, Yeo CJ, Langer R, Anderson DG, Brody JR, Sawicki
JA. CanScript, an 18-Base pair DNA sequence, boosts tumor cell-specific promoter activity. Cancer Biol
Ther. 2010 Nov 1;10(9):878-84. doi: 10.4161/cbt.10.9.13234. Epub 2010 Nov 1.
3.Garg RP, Parry RJ. Regulation of valanimycin biosynthesis in Streptomyces viridifaciens:
characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP). Microbiology. 2010
Feb;156(Pt 2):472-83. doi: 10.1099/mic.0.033167-0. Epub 2009 Nov 5.
4.Colby JK, Klein RD, McArthur MJ, Conti CJ, Kiguchi K, Kawamoto T, Riggs PK, Pavone AI, Sawicki J,
Fischer SM. Progressive metaplastic and dysplastic changes in mouse pancreas induced by
cyclooxygenase-2 overexpression. Neoplasia. 2008 Aug;10(8):782-96.
5.Weiserová M, Ryu J. Characterization of a restriction modification system from the commensal
Escherichia coli strain A0 34/86 (O83:K24:H31). BMC Microbiol. 2008 Jun 27;8:106. doi: 10.1186/14712180-8-106.
6.Ryu J, Rowsell E. Quick identification of Type I restriction enzyme isoschizomers using newly developed
pTypeI and reference plasmids. Nucleic Acids Res. 2008 Aug;36(13):e81. doi: 10.1093/nar/gkn056. Epub
2008 Jun 18.
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Costello syndrome reveals an Ang II-mediated hypertensive condition. J Clin Invest. 2008
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of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry. 2008
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Escherichia coli clinical isolates. Nucleic Acids Res. 2005 Jul 21;33(13):e114.
10.Strand SS, Leib DA. Role of the VP16-binding domain of vhs in viral growth, host shutoff activity, and
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bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI. Nucleic Acids Res. 2004 Jun
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12.Kirby AE, King ND, Connell TD. RhuR, an extracytoplasmic function sigma factor activator, is essential
for heme-dependent expression of the outer membrane heme and hemoprotein receptor of Bordetella
avium. Infect Immun. 2004 Feb;72(2):896-907.
13.Pavlova IV, Virgin HW, Speck SH. Disruption of gammaherpesvirus 68 gene 50 demonstrates that Rta
is essential for virus replication. J Virol. 2003 May;77(10):5731-9.
14.Kasarjian JK, Iida M, Ryu J. New restriction enzymes discovered from Escherichia coli clinical strains
using a plasmid transformation method. Nucleic Acids Res. 2003 Mar 1;31(5):e22.
15.Ding L, Derdowski A, Wang JJ, Spearman P. Independent segregation of human immunodeficiency
virus type 1 Gag protein complexes and lipid rafts. J Virol. 2003 Feb;77(3):1916-26.
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heme and hemoproteins. Infect Immun. 2002 Oct;70(10):5390-403.
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glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle. J Bacteriol.
2002 May;184(10):2728-39.
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recombinants in cosmids. J Virol. 2001 Jun;75