A. tumefaciens genomic DNA extraction agarose results:

FIGURE ONE ***** Results of A. tumefaciens genomic DNA extraction ***

Figure 1. GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.

In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.1 flow through, lane 3 is the DNA2.2 flow through, lane 4 is the DNA2.1 flow through and lane 4 in the DNA2.2 flowthrough. All four samples show a smear that is indicative of genomic DNA. The extraction has been successful.

Nanodrop absorbance readings:

= SUCCESS!

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon BEFORE the HO gene:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon AFTER the HO gene in the operon:

Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon AFTER the HO gene as well as the addition of a ribosome binding site or Shine Delgano sequence:

Initial PCR

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations:

FIGURE two ***** Results of the initial PCR ***

Figure 2. GelRed stained 1.2% agarose gel of initial PCR of A. tumefaciens using various primers.

In lanes 1 and 11 there is a 1kb ladder. In lane 2 is the DNA1.2 template with (AT-FWD-1) and (AT-RVS-1)primer pair.

In lane 3 is the DNA2.2 template with (AT-FWD-1) and (AT-RVS-1)primer pair. In lane 4 is the DNA1.2 template with (AT-BHO-F) and (AT-RVS-1)primer pair. In lane 5 is the DNA2.2 template with the (AT-BHO-F) and (AT-RVS-1)primer pair. In lanes 6 and 7 there is nothing loaded as the wells were damaged. In lane 8 there is the DNA1.2 template with (AT-FWD-RBS) and (AT-RVS-1)primer pair. In lane 9 there is the DNA2.2 template with the (AT-FWD-RBS)and (AT-RVS-1)primer pair and in lane 10 there is the ssH2O negative control. A product is seen in lane 9 – this is the DNA2.2 template with the (AT-FWD-RBS)and (AT-RVS-1)primers! This means that we have a A. tumefaciens bacteriophytochrome product with a ribosome binding sight inserted.

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations:

FIGURE THREE ***** PCR optimization results ***

Figure 3. GelRed stained 2% agarose gel of optimized PCR of A. tumefaciens bacteriophytochrome using various primers. In lanes 1 and 15 there is a 100bp ladder. In lane 2 is the DNA2.2 sample with (AT-FWD-1) and (AT-RVS-1)primer pair. In lane 3 there is PCR product template (from DNA2.2 sample) with (AT-FWD-1)and (AT-RVS-1)primer pair. In lane 4 there is DNA sample with (AT-BHO-F) and (AT-RVS-1)primer pair. In lane 5 is the PCR product (from DNA2.2 sample) with (AT-BHO-F)and (AT-RVS-1)primer pair. In lane 6 there is DNA2.2 sample with (AT-FWD-RBS)and (AT-RVS-1)primer pair. In lane 7 there is PCR product template (from DNA2.2 sample) with (AT-FWD-RBS)and (AT-RVS-1)primer pair. In lane 8 there is DNA2.2 sample with (AT-FWD-1) and (AT-AHO-R)primer pairs. In lane 9 there is PCR product template (from DNA2.2 sample) with (AT-FWD-1) and (AT-AHO-R)primer pairs. In lane 10 there is DNA2.2 sample with (AT-BHO-F) and (AT-AHO-R)primer pairs. In lane 11 there is PCR product (from DNA2.2 sample) with (AT-BHO-F)and (AT-AHO-R)primer pair. In lane 12 there is DNA2.2 sample with (AT-BHO-F)and (AT-AHO-R)primer pair. In lane 13 there is PCR product (from DNA2.2 sample) with (AT-FWD-RBS)and (AT-AHO-R)primer pair. In lane 14 there is ssH2O negative control with (AT-FWD-1)and (AT-RVS-1)primer pair. All the lanes worked. There is a weird result in lane 7 which is probably due to non-specific binding. = SUCCESS!

• The first primer pair is (AT-BHO-F) with (AT-BHO-R) [this will insert the bacteriophytchrome gene BEFORE the heme oxygenase gene in the operon]
• The second primer pair is (AT-AHO-F) with (AT-AHO-R) [this will insert the bacteriophytochrome gene AFTER the heme oxygenase gene in the operon]

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations and annealing temperatures:

FIGURE FOUR ***** PCR Optimisation (gradient PCR) results ***

Figure 4. No product amplification is seen in any lanes. The anticipated product is approximately 1.6 to 3.0kb. The gel had been over run and at the very bottom some bands can be seen but as these are so small they are possibly dimers that are less than 300bp.

Unfortunately, no products were seen. The anticipated product size was between 1.6kb and 3kb. The gel was over run but the only products that were over run were probably primer dimers, which are less than 300bp in size. = FAIL!

The PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 25 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

Experimental Design – Primer combinations and annealing temperatures:

The first PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 60˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 35 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.

The second PCR program was set up as per the following:

1. 94˚C for 2 minutes
2. 94˚C for 30 seconds
3. 40˚C for 30 seconds
4. 72˚C for 2 minutes & 30 seconds

(This was repeated for another 4 cycles)

5. 72˚C for 10 minutes
6. 4˚C to end.
7. 4˚C for 5 minutes
8. 94˚C for 30 seconds
9. 40˚C for 30 seconds
10. 72˚C for 2 minutes and 30 seconds

(This was repeated for another 31 cycles)

11. 72˚C for 10 minutes
12. 4 ˚C to end

Experimental Design – Primer combinations and annealing temperatures:

FIGURE FIVE ***** PCR optimization (with ssPCR amplification for (AT-BHO-F)primer) ***

Figure 5. GelRed stained 2% agarose gel of optimized PCR using primer pairs for insertion of the heme oxygenase gene and bacteriophytochrome gene. In lanes 1 and 6 there is a 1kb ladder. In lane 2 there is the (AT-FWD-RBS)-(AT-RVS-1)PCR product amplified with (AT-BHO-F)-(AT-BHO-R)primer pair on the special PCR program allowing for the (AT-BHO-R)primer to bind first. In lane 3 there is the (AT-FWD-RBS)-(AT-AHO-R)PCR product amplified with (AT-AHO-F)-(AT-AHO-R)primer pair on the normal PCR program. In lane 4 there is the (AT-FWD-RBS)-(AT-RVS-1)PCR product diluted 1:10 amplified with (AT-BHO-F)-(AT-BHO-R)primer pair on the special PCR program allowing for the (AT-BHO-R)primer to bind first. In lane 5 there is the (AT-FWD-RBS)-(AT-AHO-R) PCR product diluted 1:10 amplified with (AT-AHO-F)-(AT-AHO-R)primer pair on the normal PCR program. There is a product band seen in lane 3 – SUCCESS! This is the product with the bacteriophytochrome gene inserted AFTER the heme oxygenase gene.