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. 1. ChiP originalandChiP 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. Mini prep the plasmid, do digestion analysis:with EcoRI, PstI, EcoRV. (fig.1)
7. start of a liquid culture of transformed strain.
8. colony PCR to check if there is plasmid of correct size
9. plasmid purification
10. PCR of vector pSB1K3 which was the only choice because it is toxic to the cells so the transformation would be unsuccessful. The original template was obtained from iGEM kit 2011 and this DNA was a template for the PCR. We created also two-step PCR program specifically for the backbones. During performing PCR there was a need to add DMSO to the reaction for it to work.
11. restriction digestions of ChiP_normal:lacZ:pSB1K3, ChiP_normal:GFP:pSB1K3, ChiP_mutated:lacZ:pSB1K3 and ChiP_mutated:GFP:pSB1K3 constructs and pSB1C3 with EcoRI and PstI.
12. ligation of obtained through restriction digestion ChiP_normal:lacZ,ChiP_normal:GFP, ChiP_mutated:lacZ and ChiP_mutated:GFP with digested pSB1C3
13. transformation newly obtained constructs to E. coli NM522, E. coli IG9, E. coli IG302.
14. start of a liquid cultures of transformed strains.
15. colony PCR of this strain
16. plasmid purification and sending DNA to iGEM Headquarters
17. restriction digestion of the obtained plasmid
18. X-gel test if the plasmid is working correctly

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^o.

Project 3