Part:BBa_I742123:Experience
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UNIQ75668ccd177a21d3-partinfo-00000000-QINU UNIQ75668ccd177a21d3-partinfo-00000001-QINU
Testing the Kanamycin resistance conferred by pTG262 (BBa_I742123) in B.subtilis and E.coli
B.subtilis 168 and E. coli JM109 were each transformed with the vector pTG262 (BBa_I742123) with an RFP biobrick insert cassette.
B.subtilis
WT B. ubtilis 168 and pTG262 transformed B.subtilis 168 colonies were used to inoculate 5ml LB medium in glass standard bottles which were incubated at 37C with shaking and loose lids for ~6h (pTG262 with 5mg.L-1 chloramphenicol to prevent vector loss). This culture was then diluted by a factor of 10-6 and 100ul was plated onto LB agar plates of varying kanamycin concentrations, and incubated overnight at 37C.
The number and size of colonies which grew on each of the agar plates was recorded.
E.coli test of growth on agar plates
WT E.coli JM109 and pTG262 transformed E.coli JM109 colonies used to inoculate 5ml LB medium in glass standard bottles which were incubated at 37C with shaking for 6h30m (pTG262 inoculated in duplicate with no antibiotic and 10mg.L-1 Kanamycin).
The OD600 of the cultures was measured:
Each of the above cultures were diluted by factors of 10-5 and 10-6. Agar plates of varying kanamycin concentration were plated with 100ul aliquots of the dilutions, and incubated for ~36h at 37C. The number and colour of colonies was recorded (red indicating presence of pTG262, white being WT or representing loss of the plasmid before plating).
Figure 1. A plate from the above experiment after an additional 24 hours on the bench, pTG262 Kan0 10-5 dilution plated onto 0mg.L-1 Kanamycin. This shows the typical colony size as indicated in the above table. It demonstrates both presumed plasmid loss in overnight culture without antibiotic (white colonies) and the decreased growth rate of cells containing pTG262.
E. coli test of growth in Kanamycin liquid culture
Overnight LB liquid cultures of WT E.coli JM109 and E.coli JM109 transformed with pTG262 were diluted 1:50 in fresh LB medium. 3ml aliquots of each were put in glass standard bottles, along with varying amounts of kanamycin. The Optical density at 600nm was measured at the start and after 8 hours incubation with shaking at 37C.
Initial OD600: WT JM109 - 0.030, pTG262 - 0.028.
OD600 after 8 hours incubation with shaking at 37C:
BBa_I742123 derekju -MIT iGEM 2008 |
We worked extensively with BBaI742103 and BBaI742123 trying to transform it into Lactobacillus delbruckii subsp. bulgaricus, Lactobacillus delbruckii subsp. lactis, and Lactobacillus acidophilus. The DNA from the 2008 registry failed to transform and Dr. French, who entered this part, was kind enough to supply us with the plasmids. It turned out the registry wasn't able to transform this plasmid into E. Coli, probably due to non-typical growth conditions. Instructions to transform into E. Coli:
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No review score entered. alex273, Lyon-INSA 2012 |
We found an additional PstI site, which prevented us from doing any cloning in this vector. As, to our knowledge, this was the only available and working shuttle vector for B.subtilis and E.coli, we built our own plasmid, with a low-copy (K802003) or high-copy (K802004) version. These new plasmids were successfully transformed in B.subtilis and E.coli, no other organism was tested. A full characterization is available on their main pages, including antibiotic resistance thresholds and transformation efficiencies. |