Non-antibiotic-resistant vectors for microprojectile-mediated transformation of plants
Last modified July 23, 2001; information still current October 2004

What are pMECA-R and pBluescript-R?

These are cloning vectors in which the gene for ampicillin resistance has been replaced with most of an operon for ribitol utilization, derived from E. coli strain C.

Medium requirements

• These plasmids permit growth when ribitol is the sole carbon source. Hence,the use of a defined medium is required. We have experienced the best success with 2B Minimal Medium, supplemented with 2 g/L of ribitol:

Stock I (Use 100 mL per L) NH4Cl – 2 g per 100 mL KH2PO4 – 6 g per 100 mL Na2HPO4 – 12 g per 100 mL

Stock II (Use 50 mL per L) MgSO4 7H2O – 0.26 g per 100 mL

Stock III (Use 20 mL per L) CaCl2 2H2O – 0.37 g per 100 mL

Note as well that laboratory K-12 strains all require other supplements for growth, with each strain requiring its own combination. The necessary supplements for some strains are as follows:

• DH10B – 1 mg/L of thiamine and 50 mg/L each of L-leucine and L-isoleucine DH5alpha- 50 mg/L each of of L-leucine and L-isoleucine
• TOPTEN – 50 mg/L of L-leucine

Potential limitations:

• The observation that XbaI digests pMECA-R 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.

Slow growth

Expect the cultures to take 48 hrs to reach log phase in culture.

What is the reference for pMECA-R and pBluescript-R?

• LaFayette, P.R. and W.A. Parrott. 2001. A non antibiotic marker for amplification of plant transformation vectors in E. coli. Plant Cell Rep. 20:338-342. PDF.

Where can I find the sequence for the ribitol operon?

GenBank # AY005817. The repressor sequence is not available.

Are there any restrictions on the use of pMECA-R and p-Bluescript-R?

These plasmids are being made available on a non-exclusive basis for non-commercial research uses, with the following conditions:

• These plasmids will not be transferred to any third parties without permission from their originators Any publications resulting from the use of these plasmids will acknowledge their use and cite the appropriate reference No guarantees, explicit or implied, on the effectiveness or efficiency of either plasmid, are made by their originators or The University of Georgia, nor can their originators or The University of Georgia be held liable for failure of the plasmids to perform as expected, or any consequences resulting there from Ultimately, it will be up to the various regulatory agencies to determine if it will be acceptable for vector sequences from these plasmids to become incorporated into a transgenic organism. As of yet, no such determination has been made.
• Anyone desiring to use either plasmid for any commercial purpose must contact Shelley Fincher at The University of Georgia Technology Transfer Office for additional information.