A: A better way to test for sucrose sensitivity is to patch individual colonies from a LB + Kan (40 mg/ml) plant onto a LB + Kanamycin (40 mg/L) master, then replica plate (print) to a Kan + sucrose plate – this gives an accurate result. If, for example you streak out DH10B (BIBAC2) onto Kan + Sucrose you will always get some colonies – there is a very strong selection pressure to survive. Most likely the cells that survive will have deletions or rearrangements in the sacB gene. A related problem is: when constructing libraries there will always be some background, that is, vectors without inserts that appear on Kan + sucrose plate. This is true for lacZ, sacB and essentially any marker system.
Frequently Asked Questions:
Q: I plated out DH10B + BIBAC2 vector on Kan (40 mg/L) + Sucrose (5%) plates and got some colonies. What is going on?
A: You should use whatever A. tumefaciens strains performs best for your plant system. The BIBAC plasmids work well with a variety of A. tumefaciens strains (Hamilton et al. 1997).
Q: We use “supervirulent” Agrobacterium strain EHA101 (or EHA105) for plant transformation. Can we use a BIBAC vector in these strains?
A: Any A. tumefaciens strain is fine as long as it is not (already) resistant to kanamycin – since BIBAC plasmids carry the gene for resistance to kanamycin as the selectable marker. EHA101 contains a disarmed pTi that is carries resistance to kanamycin thus, is not suitable for use in conjunction with the BIBAC vectors. Also, our experience was that recA- strains performed better for high molecular weight DNA transformation – although this may not always be the case.
Q: Do you use different protocols for restriction, ligation, etc.) for a BIBAC than for other plasmids?
A: There are many cloning “styles” – all of which can be successful. This site includes protocols for subcloning and modification of the BIBAC etc.. For library construction protocols go to the Texas A&M BAC Center site. BIBAC library construction details will provided at this site at a later date.
A: No, use the plant transformation protocol developed for your plant system. Be sure to use glucose instead of sucrose for co-cultivation and subsequent tissue cultures steps for BIBAC vector (without insert) control experiments – as the sacB gene also functions in A. tumefaciens, and will kill the A. tumefaciens cells when plated on media containing high sucrose.
Q: NPTII is not a good marker for selecting transformants in our plant system. Can we use BIBAC2 and select for HYG?
A: You could, but you probably don’t want to. The marker that is used to initially select for transformants should be located adjacent to the left T-DNA border (LB) since the T-DNA is transferred right border (RB) to left border. If you use BIBAC2 and select for hygromycin resistant transformed plant tissue it is likely (although this may vary depending on the plant system) that the T-DNAs transferred will not be complete/intact.
A: Maybe not. However the larger your insert is the more likely it is that it will contain some “problem sequences”. If you use the recombination deficient recA- strain of A. tumefaciens you improve your chances of maintaining problem inserts. In some plant systems the recA- A. tumefaciens strains do not perform as well as the isogenic recA+ strains for transfer of a BIBAC vector alone (~ 7 kb T-DNA). However, for the 150 kb test construct the recA- strains gave a higher efficiency of transfer. Additional work is in progress. We really don’t have enough data at this point to make a strong statement concerning recA- strains – but I still recommend it.
A: Although any particular protocol may work – we have included what has worked best in our hands.
Q: The existing BIBAC vectors only have BamHI as a unique site for library construction. Why don’t you put in some other sites by site-directed mutagenesis, or perhaps a polylinker?
A: The BIBAC vectors are big. It was difficult to maintain the unique BamHI site. The BamHI site is also compatible is BglII and Sau3A. Other sites that are unique to the BIBAC backbone pCH20 are SrfI, SwaI, PacI and AscI. One (newly available) enzyme that does not cut the existing BIBAC vectors is ApaLI. You can assume that all other commercially available enzymes (6/98) cut the BIBAC vectors at least once.
A: No. We recommend CsCl preps of BIBAC plasmids to obtain quantities of high quality DNA.
A: No. The BIBAC vectors use the Ri origin for replication in A. tumefaciens. Thus, the existing BIBAC vectors are incompatible with the pRi plasmid in A. rhizogenes. However, if the minimal Ti origin was substituted for the minimal Ri origin then the resulting BIBAC would be compatible with A. rhizogenes.
A: No. But as soon as it is – we will post it here.
A: Yes. We made two tomato libraries with average insert sizes of 100 and 125 kb (manuscript submitted). I have also heard that other groups have successfully constructed BIBAC libraries.