Davidson College Department of Biology Honors Thesis Title ...

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Figure 16: Integration plasmid pG80ko with LuxR/LasR expression cassette (BBa K091206). The R6K origin requires Pir expression for replication. Genomic integration occurs via a recombination event between the plasmid’s att80 site and the Φ80 site on the E. coli chromosome. The plasmid is insulated with two transcriptional terminators to prevent read-through transcription once in the genome. Figure 17: Helper plasmid pInt80-649. When present in the cell, the helper plasmid expresses pir and allows for replication of pG80ko. When exposed to 43 ◦ C temperatures, CI857 becomes inactive and pInt80-649 cannot be replicated. Curing cells of the helper plasmid also cures them of the insertion plasmid. Therefore, only cells that have undergone a genomic insertion can survive gentamicin selection at 43 ◦ C. 24
and cells that have not undergone integration lose their resistance to gentamicin. At 43 ◦ C, only transgenic cells should express a gentamicin resistance gene. Genomic integration is verified by PCR near the Φ80 integration site on the E. coli chromosome. As specified by Haldimann and Wanner (2001), 4 primers allowed me to distinguish between a single and multiple integration events (Materials and Methods, Table 2). I used this basic framework to integrate K091206 into the E. coli genome. First, I needed to clone K091206 into pG80ko using an EcoRI/PstI restriction digest and ligation. Unfortu- nately, the difference in fragment length between K091206 and the pSB1A2 vector in which it resided was only 39 bps when digested with EcoRI/PstI. This difference was too small to resolve on a gel, as can be seen in Figure 15. In order to move K091206 into pG80ko, I searched for a unique restriction site somewhere in the middle of pSB1A2 vector. Luckily, there was a ScaI site and by doing a triple digestion of the pSB1A2-K091206 plasmid (EcoRI, ScaI, and PstI), I was able to cut the vector into 1492 bp and 550 bp fragments. The bands resolved nicely once the vector was cut in two pieces (Figure 18). I transformed the ligation of K091206 and pG80ko into Ec100D::pir+ cells that had been sent from the Anderson Lab at UC Berkeley. These cells constitutively expressed pir protein and made replication of the pG80ko plasmid possible. I picked two colonies from the transformation and verified for successful ligation (Figure 19). The final step to the genomic insertion was the cotransformation of the helper plasmid, pInt80-649, and the integration plasmid into the target strain. The paper by Haldimann and Wanner indicated a wide range of strains were suitable for this method. We chose HB101 cells as the strain to receive the genomic insertion because another student in the lab was working on a different project in this cell strain and it does not have a LacI gene which might affect future projects. The transformations were performed in succession, with the helper plasmid, pInt80-649, being transformed in first so that pir expression could begin before 25
Page 1 and 2: Davidson College Department of Biol
Page 3 and 4: Introduction Synthetic Biology Rece
Page 5 and 6: function in vivo, but also to a lac
Page 7 and 8: Figure 2: Structure of AHL autoindu
Page 9 and 10: are used to obtain quick and easy a
Page 11 and 12: mations, satellite colonies are unw
Page 13 and 14: Figure 6: BioBrick Standard Assembl
Page 15 and 16: (http://www.bio.davidson.edu/course
Page 17 and 18: trol promoter, pLas’, were constr
Page 19 and 20: (a) LacII12X86-pLuxLas (b) LacIX86-
Page 21 and 22: my negative control plate (no GFP i
Page 23: Figure 15: EcoRI and PstI digestion
Page 27 and 28: transformation of the integration p
Page 29 and 30: When I picked one of the MC4100 col
Page 31 and 32: Figure 23: GFP expression of pLas
Page 33 and 34: Figure 25: GFP expression of the Lu
Page 35 and 36: Measuring Time-delayed Growth In ad
Page 37 and 38: Figure 29: Distance of non-resistan
Page 39 and 40: Figure 31: Distance of non-resistan
Page 41 and 42: assumption that repression would ov
Page 43 and 44: might be the culprit since it is ex
Page 45 and 46: Regardless, the linear data made it
Page 47 and 48: D. Ferber. Synthetic biology. micro

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