Constructed plasmids

P_ChiP-lacZ fusion

The plasmids containing this DNA fragments were obtained by the following procedure:

1. PCR of:
   1. upstream part of ChiP using E. coli w3110 genomic DNA as a template and TAQ enzyme. Using two different downstream primers (one wild type and one with mutation). we obtained two types of ChiP: original and mutated, which has altered binding site for ChiX.
   2. lacZ genefrom BBa_I72005 biobrick using phusion enzyme.
2. Restriction digestions of:
   1. ChiP original and ChiP mutated with XbaI and NcoI,
   2. lacZ gene with BspHI and PstI
   3. GFP gene with BspHI and PstI
   4. pSB1C3 with EcoRI and PstI
3. ligations of:
   1. ChiP normal with lacZ and pSB1C3
   2. ChiP mutated with lacZ and pSB1C3
4. transformations to E. coli NM522 cells.
5. plating the bacterialand grow over night in 37℃ incubator
6. start of a liquid culture of transformed strain.
7. Mini prep the plasmid, do digestion analysis:with EcoRI, PstI, EcoRV. (fig.1)
8. Transformation to NM522, IG9,IG302.
9. 'β-gal assay

P_Tet-chiX fusion

The plasmids containing this DNA fragments were obtained by the following procedure:

1. start liquid culture of minigenes: chiX, chiXR, tetR
2. PCR:pSB4k5 (or any other non cam non pSB1 pSB6 plasmid)
3. purification of the PCR product
4. mini prep minigenes
5. Restriction digestions:
   1. chiX with XbaI and PstI
   2. tetR with EcoRI and SpaI
   3. pSB1C3 with EcoRI and PstI
   4. pSB4k5 with EcoRI and PstI
6. ligations of:tetR with chiX and pSB1C3
7. transformations to E. coli NM522 cells.
8. plating the bacterialand grow over night in 37℃ incubator
9. start of a liquid culture of transformed strain.
10. Mini prep the plasmid, do digestion analysis:with EcoRI, PstI (fig.2)
11. subclone Ptet-chiX part into pSB4k5
12. transformation to E. coli NM522 cells.
13. Colony PCR with CS149/150 on transformants.Pick 4 positive colonies and grow overnight
14. Minipreps the corect ones, Transform into IG302/pSB1C3 PchiPlacZ
15. Beta-Galactosidase Assay Pick 2 transformants and grow. Make Beta-Galactosidase Assay +/- atc.

Strain construction

The aim of this experimental section was to create strain with following gene deletetions:lacZYA and chbBCARG operon.

1. PCR of recombineering linear pieces of DNA using pSLD31 plasmid and lacZ upstream/downstream primers pair and chb upstream/downstream primers pair. These PCR products contained kanamycin resistance gene.
2. Transformation of the E. coli W3110 strain containing pSLD18 plasmid (provides Red recombineering system) with pfhc3220 plasmid (containig Cre recombinase).
3. Once the strain containing both plasmids was constructed we were ready to proceed with the first round of recombineering. We followed the protocal describing this procedure to achieve first gene deletion - $\Delta$chbBCARG. First we screened for the gain of kanamycin resistance and did the colony PCR to confirm orrect insert size. Then the Cre recombinase was activated and facilitated pop-out event the resistance marker. We screened for loss of resistance and, again, confirmed the correct sizes with colony PCR. Colony PCRs were done using CSchb upstream/downstream primers pair. Correct strain was saved as IG7 and stored under -80^oC.

Project 3