Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download ConjugationSlides
Document related concepts
Artificial gene synthesis wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Genetic engineering wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
DNA vaccination wikipedia , lookup
List of types of proteins wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Transcript
Bacterial Conjugation • Conjugation will be used as a means to transfer genetic information from one bacteria to another • Cells with a F+ plasmid or a R plasmid can transfer their DNA to other bacteria • On the F and R plasmids, DNA transfer begins at a specific sequence, OriT, the Origin of Transfer. •OriT can be knocked out to severely hinder the efficiency of conjugation, but the efficiency can be restored by a complementation in trans. 1 OriT Complementation Lock OriT OnGFP Key OriT Key OriT 2 Conjugation Assays Matings with recipient cell ec100D::pir+, which is TriR, show if the F or R plasmid transfers by the TriK plate and if an OriT plasmid transfers by the TriA plate Wildtype F plasmid pOX38 pOX38 x pSB1A2 - J01064 J23015 J23015 x pSB1A2 - J01064 Wildtype R plasmid RP1 Rlambda Rlambda x pSB1A2 - J01003 OriT cis cis&trans none trans # of Colonies on TriK 396800 288000 0 1000 # of Colonies on TriA n/a 176000 0 126400 cis none trans 112000 1 0 n/a 0 1664 Mutation and complementation of oriT for both F and R plasmids works! 3 Sending the Key to the Lock Mating Results of sending the key plasmid to a cell holding the locked RFP plasmid, pJ23006-J23077 pJ23006-J23086 x BW20767 pJ23006-J23066 x BW20767 pSB1A2 - J01003 x BW20767 TG1 OriT trans none trans none Key yes yes no no # of Colonies on AmpCam 109600 3 140800 0 Mating Results of sending the key plasmid to a cell holding a OnRFP plasmid, pSB1A2-J01022 pJ23006-J23086 x BW20767 pJ23006-J23066 x BW20767 pSB1A2 - J01003 x BW20767 TG1 trans none trans none yes yes no no 160000 4 147200 0 Assay shows the key can be transferred efficiently to the lock! 4 Tecan Fluorescence Assay The Tecan test shows that the key plasmid results in the recovery of fluorescence mated with J23077 503 74 pJ23006-J23086 x BW20767 pSB1A2 - J01003 x BW20767 mated with J01022 4745 5913 The controls show that conjugation is equivalent to a cotransformation of the key and lock plasmids into a cell pJ23006-J23086 x BW20767 (transformed in TGIs) TGI pJ23006 - J23077 473 61 63 7000 6000 5000 4000 3000 2000 Series1 Graph will NOT BE USED Series2 1000 pJ23006 J23077 pJ23006J23086 x BW20767 (transformed pJ23006J23086 x BW20767 (mated with 0 pJ23006J23086 x BW20767 (mated with RFP Flurorescence Graph 5
