Team:Macquarie Australia/Notebook
From 2010.igem.org
PROJECT LAB BOOK
Welcome to the Macquarie University project lab book page!
Here you will find a day-by-day account of our triumphs and failures.
A day-by-day progress for Agrobacterium Tumefaciens Bacteriophytochrome
Genomic DNA extraction
Figure 1. GelRed stained 1% agarose gel of genomic DNA extraction from A. tumefaciens.
In lane 1 there is a 1kb ladder. In lane 2 is the DNA1.1 flow through, lane 3 is the DNA1.2 flow through, lane 4 is the DNA2.1 flow through and lane 5 is the DNA2.2 flowthrough. All four samples show a smear that is indicative of genomic DNA. The extraction has been successful.
Nanodrop absorbance readings:
Genomic DNA sample | 260/280 OD ratio | Concentration (ng/mL) |
---|---|---|
DNA1.1 | 1.88 | 351.5 |
DNA1.2 | 1.98 | 203.1 |
DNA2.1 | 2.14 | 738.9 |
DNA2.2 | 2.16 | 709.7 |
= SUCCESS!
27th August 2010
Primer design
Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene:
Primer name | Primer Sequence |
---|---|
(AT-FWD-1) | 5’-ATG AGT TCA CAT ACG CCG-3’ |
(AT-RVS-1) | 5’-TCA GGC AAT TTT TTC CTC-3’ |
Fwd and Rvs primers for amplification of the full length A. tumefaciens BphP gene for insertion in the operon BEFORE the HO gene:
Primer name | Primer Sequence |
---|---|
(AT-BHO-F) | 5’- AAG GAG ATA TAC ATA TGA TGA GTT CAC ATA CGC CG – 3’ |
(AT-BHO-R) | 5’- AAG TTG ACA CTC ATA TGA GCC CTC CTT TCA GGC – 3’ |
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:
Primer name | Primer Sequence |
---|---|
(AT-AHO-F) | 5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG – 3’ |
(AT-AHO-R) | 5’- GTT AGC CGG ATC CTC AGG CAA TTT TTT CCT – 3’ |
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:
Primer name | Primer Sequence |
---|---|
(AT-FWD-RBS) | 5’- AGG AGG GCT ATG AGT TCA CAT ACG CCG -3’ |
Initial PCR
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations:
Template DNA | Fwd Primer | Rvs primer |
---|---|---|
DNA1.2 | (AT-FWD-1) | (AT-RVS-1) |
DNA2.2 | (AT-FWD-1) | (AT-RVS-1) |
DNA1.2 | (AT-BHO-F) | (AT-RVS-1) |
DNA2.2 | (AT-BHO-F) | (AT-RVS-1) |
DNA1.2 | (AT-FWD-RBS) | (AT-RVS-1) |
DNA2.2 | (AT-FWD-RBS) | (AT-RVS-1) |
SSH20 (negative control) | (AT-FWD-1) | (AT-RVS-1) |
= SUCCESS!!!
= SUCCESS!!
Figure 2. Results of the initial PCR
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.
PCR Optimization
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations:
Template DNA | Fwd Primer | Rvs primer |
---|---|---|
DNA2.2 | (AT-FWD-1) | (AT-RVS-1) |
PCR product (from DNA2.2) | (AT-FWD-1) | (AT-RVS-1) |
DNA2.2 | (AT-BHO-F) | (AT-RVS-1) |
PCR product (from DNA2.2) | (AT-BHO-F) | (AT-RVS-1) |
DNA2.2 | (AT-FWD-RBS) | (AT-RVS-1) |
PCR product (from DNA2.2) | (AT-FWD-RBS) | (AT-RVS-1) |
DNA2.2 | (AT-FWD-1) | (AT-AHO-R) |
PCR product (from DNA2.2) | (AT-FWD-1) | (AT-AHO-R) |
DNA2.2 | (AT-BHO-F) | (AT-AHO-R) |
PCR product (from DNA2.2) | (AT-BHO-F) | (AT-AHO-R) |
DNA2.2 | (AT-FWD-RBS) | (AT-AHO-R) |
PCR product (from DNA2.2) | (AT-FWD-RBS) | (AT-AHO-R) |
ssH2O (-) control | (AT-FWD-1) | (AT-RVS-1) |
Figure 3. PCR optimization results
PCR Optimization (using Gradient PCR)
- 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]
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations and annealing temperatures:
Fwd Primer | Rvs primer | Temp 1 (Degrees Celsius) | Temp 2 (Degrees Celsius) | Temp 3 (Degrees Celsius) | Temp 4 (Degrees Celsius) | Temp 5 (Degrees Celsius) |
---|---|---|---|---|---|---|
(AT-BHO-F) | (AT-BHO-R) | 57.1 | 58.7 | 60.6 | 63.4 | 64.8 |
(AT-AHO-F) | (AT-AHO-R) | 57.1 | 58.7 | 60.6 | 63.4 | 64.8 |
Figure 4. PCR Optimisation (gradient PCR) results
No products were observed. 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!
PCR Optimization (using Gradient PCR)
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations and annealing temperatures:
DNA template | Dilution | Fwd primer | Rvs primer | Annealing temp (Degrees Celsius) |
---|---|---|---|---|
PCR product (from DNA2.2) | 1:100 | (AT-BHO-F) | (AT-BHO-R) | 60 |
PCR product (from DNA2.2) | 1:200 | (AT-BHO-F) | (AT-BHO-R) | 60 |
PCR product (from DNA2.2) | 1:100 | (AT-AHO-F) | (AT-AHO-R) | 60 |
PCR product (from DNA2.2) | 1:200 | (AT-AHO-F) | (AT-AHO-R) | 60 |
PCR product (from DNA2.2) | 1:100 | (AT-BHO-F) | (AT-BHO-R) | 65 |
PCR product (from DNA2.2) | 1:200 | (AT-BHO-F) | (AT-BHO-R) | 65 |
PCR product (from DNA2.2) | 1:100 | (AT-AHO-F) | (AT-AHO-R) | 65 |
PCR product (from DNA2.2) | 1:200 | (AT-AHO-F) | (AT-AHO-R) | 65 |
PCR Optimization (repeated)
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The first PCR program was set up as per the following:
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
(This was repeated for another 35 cycles)
The second PCR program was set up as per the following (this was for the amplification of product using the (AT-FWD-RBS) and (AT-AHO-R) primer pair):
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 40˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 10 minutes
- 4˚C to end.
- The second PCR program was set up as per the following. There were two parts to this PCR to allow for the (AT-BHO-F) primer to anneal properly after initial annealing of the (AT-BHO-R) primer.
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 40˚C for 30 seconds
- 72˚C for 2 minutes and 30 seconds
- Now we can add the (AT-BHO-F) primer to the reaction.
- 4˚C for 5 minutes
- 94˚C for 30 seconds
- 60˚C for 30 seconds
- 72˚C for 2 minutes and 30 seconds
- 72˚C for 10 minutes
- 4˚C to end
- The PCR products were run on a GelRed post-stained 2% agarose gel using a 1kb ladder for visualization
- Various primers were designed manually and using Primer 3 Software package for PCR amplification.
- The primers were ordered and supplied through Integrated DNA Technologies.
- There was an array of various primers ordered for amplification of different products. The details of the primers are described below.
- The reaction mastermix for the PCR was set up as per the following recipe (per sample):
- 94˚C for 2 minutes
- 94˚C for 30 seconds
- 55-65˚C for 30 seconds
- 72˚C for 2 minutes & 30 seconds
- 72˚C for 5 minutes
- 4˚C to end.
- Different combinations of the primers were used for the initial PCR reaction (see below ‘Experimental Design’ section following)
- Not all possible primer combinations were used
- The PCR products were run on a 2% GelRed post-stained agarose gel for visualisation (see picture of gel below).
- All 85 reactions were unsuccessful and no bands were visible on the gel = BACK TO THE DRAWING BOARD!
(This was repeated for another 35 cycles)
(This was repeated for another 4 cycles)
(This was repeated for another 31 cycles)
Experimental Design – Primer combinations and annealing temperatures:
Template | Primer pair | Annealing temp (degrees Celsius) | Number of cycles |
---|---|---|---|
(AT-FWD-RBS)-(AT-RVS-1)PCR product | (AT-BHO-F)-(AT-BHO-R) | 40, then 60 | 4, then 35 |
(AT-FWD-RBS)-(AT-AHO-R)PCR product | (AT-AHO-F)-(AT-AHO-R) | 60 | 35 |
(AT-FWD-RBS)-(AT-RVS-1)PCR product (1:10 dilution) | (AT-BHO-F)-(AT-BHO-R) | 40, then 60 | 4 then 35 |
(AT-FWD-RBS)-(AT-AHO-R)PCR product (1:10 dilution) | (AT-AHO-F)-(AT-AHO-R) | 60 | 35 |
Figure 5. PCR optimization (with ssPCR amplification for (AT-BHO-F)primer)
Discussion of gradient PCR:
The gradient PCR was used because the (AT-BHO-R) primer that was ordered was 12 base pairs shorter than what was supposed to be ordered. The (AT-BHO-R)primer was supposed to include the whole reverse primer sequence (18 base pairs) as well as an additional sequence for the HO site. This caused an issue with this primer annealing in previous PCR reactions. The gradient PCR will allow for this primer to anneal prior to any other primer annealing increasing the chance of annealing.
A day-by-day progress for Deinococcus Radiodurans Bacteriophytochrome Operon Construct
Genomic DNA extraction
Figure 6. GelRed post-stained 1% agarose gel of genomic DNA extraction from D. radiodurans . In lanes 1 and 5 there is a 1kb ladder. In lane 2 is the DEINO1 sample, lane 3 is the DEINO2 sample, lane 4 is the DEINO3 sample. All three samples show a smear that is indicative of genomic DNA. The extraction has been successful!!!
Nanodrop absorbance readings:
Genomic DNA sample | 260/280 OD ratio | Concentration (ng/mL) |
---|---|---|
DEINO1 | 2.43 | 1.443 |
DEINO2 | 2.55 | 51.8 |
DEINO3 | 2.99 | 88.7 |
= SUCCESS!
27th August 2010
Primer design
Fwd and Rvs primers for amplification of the full length D. radiodurans BphP gene:
Primer name | Primer Sequence |
---|---|
(DR-FWD-1) | 5’-ATG AGC CGG GAC CCG TTG -3’ |
(DR-RVS-1) | 5’-TCA GGC ATC GGC GGC TCC -3’ |
Fwd and Rvs primers for amplification of the full length D. radiodurans BphP gene for insertion in the operon BEFORE the HO gene:
Primer name | Primer Sequence |
---|---|
(DR-BHO-F) | 5’- AAG GAG ATA TAC ATA TGA TGA GCC GGG ACC CGT TG – 3’ |
(DR-BHO-R) | 5’- AAG TTG ACA CTC ATA TGA GCA GCC CTC CTT CAG GC – 3’ |
Fwd and Rvs primers for amplification of the full length D. radiodurans BphP gene for insertion in the operon AFTER the HO gene in the operon:
Primer name | Primer Sequence |
---|---|
(DR-AHO-F) | 5’- CCG AAG GCT AGG ATC CAG GAG GGC TGC TAT GAG C – 3’ |
(DR-AHO-R) | 5’- GTT AGC AGC CGG ATC CTC AGG CAT GGG CGG CTC C – 3’ |
Fwd and Rvs primers for amplification of the full length D. radiodurans 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:
Primer name | Primer Sequence |
---|---|
(DR-FWD-RBS) | 5’- AGG AGG GCT GCT ATG AGC CGG GAC CCG TTG -3’ |
Initial PCR (gradient PCR)
Mastermix: | Amount per sample (ul) |
---|---|
Gibco H2O | 13.75 |
10x Buffer | 2.00 |
Polymerase enzyme | 0.25 |
dNTP | 1.00 |
Fwd primer | 1.00 |
Rvs primer | 1.00 |
Genomic DNA | 1.00 |
Total | 20.00 |
The PCR program was set up as per the following:
(This was repeated for another 25 cycles)
Experimental Design – Primer combinations and annealing temperatures:
DNA template | Dilution | Fwd primer | Rvs primer | Annealing temp (Degrees Celsius) | |
---|---|---|---|---|---|
DEINO1 | 1:20 | (DR-FWD) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:20 | (DR-FWD) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:20 | (DR-FWD-RBS) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:20 | (DR-FWD-RBS) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:20 | (DR-FWD) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:20 | (DR-FWD) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:20 | (DR-FWD-RBS) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:20 | (DR-FWD-RBS) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:50 | (DR-FWD) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:50 | (DR-FWD) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:50 | (DR-FWD-RBS) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:50 | (DR-FWD-RBS) | (DR-RVS) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:50 | (DR-FWD) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:50 | (DR-FWD) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO1 | 1:50 | (DR-FWD-RBS) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
DEINO2 | 1:50 | (DR-FWD-RBS) | (DR-AHO-R) | 55, 57, 60, 62, 65 | |
(-) control | - | (DR-FWD) | (DR-RVS) | 55, 57, 60, 62, 65 |
PCR with FailSafeTM PCR system