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Team:Westminster/Results
From 2012.igem.org
WIKI Notebook & Results
PROJECT DESIGN: WEEK 1 (16 July 20120
WEEK 2 This week a protocol for identifying the promoter sequences of the ALDH isoforms has been designed.
WETLAB WEEK 1 (12 September 2012) After months of planning we are finally in the lab!
Wednesday AIM: The primers for
amplification of the ALDH promoter sequences have arrived. These primers have
the Biobrick overhangs. Primers were diluted to 100mM
and working concentrations of primers was made up at 10mM. The first set of
primers to amplify the four ALDH isoforms was set up. PCR was set up using Pfu polymerase. Template used was whole cells (mammalian). Cells were thawed, spun
down and the supernatant removed and re-suspended in 50uL DMSO. We attempted to
amplify the following promoter sequences: ALDH1A1, ALDH2, ALDH1A3, ALDH3A1 (1)
and ALDH3A1(2). The cycle conditions were as follows: Initial denaturation at 980C for 30 seconds then
30 cycles with 980C for 10 seconds, 540C for 30 seconds
(annealing) and 720C for 30 seconds. Final extension was at 720C
for 10 minutes and then PCR was held at 40C. RESULTS: No bands were seen
on the gel. ANALYSIS: It is possible that
the DNA template (addition of whole cells) did not become available in the
reaction. Other cellular components during lysis of
the cells may also have had an impact on the efficiency of the PCR reaction. We
have located a protocol for crude extraction of mammalian DNA and will repeat
the PCR using extracted DNA. The extraction protocol was performed. (see protocols) Sample 1 was used as DNA template in the next PCR reaction. Nanodrop readings were as follows: Sample 1: 42.1ng/µL
with purity ration 260/280 = 1.47 Sample 2: 50.4ng/ µL with purity ration 260/280 = 1.30 Sample 3: 46.4ng/ µL with purity ration 260/280 = 1.28 Thursday AIM: Amplification of ALDH
promoters. A PCR reaction was carried out as before with the same reaction mix
and PCR conditions as Wednesday. amplify ALDH1A1 and
ALDH2. RESULTS: Amplification of ALDH1A1 was
successful using the ALDH1A1-Fw and ALDH-Rv primers.
The band size was as expected. ALDH 2 was also amplified using ALDH2-Fw and
ALDH2-Rv primers. ANALYSIS: We are pleased with developments thus
far. Difficulties in amplification of ALDH3A1 using our the ALDH3A1(1)
and ALDH3A1(2) primers was anticipated. Initial analysis in selecting an
ALDH3A1 promoter region revealed a number of different options. Additionally,
it was difficult to find commercial sequences which matched the gene sequence
identified in NCBI. Subsequent to ordering the primers, the Eukaryotic Promoter
Database was discovered. Primers for the EPD experimentally designed ALDH3A1
promoter have been ordered and will be tested when they arrive. ALDH1A1 was initially designed to be
quite large and also includes an illegal site. An experimentally tested ALDH1A1
promoter has been identified on EPD. There is significant overlap between this
EPD promoter sequence and the one we had designed. A new forward EPD designed
primer was ordered which would give a smaller promoter and also eliminate the
illegal site. Friday We have decided to try a touchdown
PCR reaction in order to try to amplify the other parts, particularly ALDH1A1.
Touchdown PCR is a faster but a more crude method of amplification in
comparison to gradient PCR. After setting up the first PCR, we realised we had
made a mistake (Touchdown 1). A second touchdown (Touchdown 2) was set up and
run soon after with the corrections. We managed to get bands for all four
promotes. These were cut from the gel, gel extracted, cut with EP (and ES for
ALDH2), ligated and transformed into competent E coli. Touchdown 1 cycle conditions were as follows: Initial denaturation at 980C for 30 seconds then
10 cycles with 980C for 10 seconds, 620C for 30 seconds
(annealing) and 720C for 1 minute 10 seconds, and annealing
temperature decreasing by 10C for each cycle. Second amplification
cycle was 20 cycles of 980C for 30 seconds, 520C for 10
seconds (annealing) and 720C for 1 minute 10 seconds. Final
extension was at 720C for 10 minutes and then PCR was held at 40C.
NOTE: In the second cycle (20 cycles) denaturation time at 980C
should have been 10 seconds and annealing time should have been 30 seconds.
This correction was included in Touchdown 2. Additionally, DNA template
concentration was increased in Touchdown 2, by increasing the volume from 1µL per reaction to 5 µL per reaction RESULTS: Spurious bands were
obtained in the touchdown 1, but ALDH1A1 and ALDH2 were amplified. Touchdown 2 gave good results for ALDH2.
For ALDH 1A3 and number of bands were seen. A faint band was observed at the
expected size and this is the band which was cut from the gel. ALDH1A1 from gel
1, ALDH2 from gel 2 and ALDH3A1 from gel 2 were also cut from the gel and gel
purified. Extracted DNA was run on a gel to verify the gel extraction process. Gel extracted DNA was digested with EP
and ALDH2 was restricted with ES. The backbone was restricted with EP and ES aswell. Restriction digests were saved till the next day. GEL 1-Touchdown 1 Gel 2-Touchdown 2 WEEK 2 Monday The linker primers and the EPD primers have arrived today. Touchdown PCR to amplify the promoter
parts was set up. The touchdown PCR was as before. The forward ALDH1A1(EPD) primer was paired with the reverse primer
ALDH1A1-RV. The EPD designed primers for ALDH3A1 were tested. RESULTS: It worked a dream!
We have got lovely bands for ALDH1A1(EPD), ALDH2 and
ALDH3A1 (EPD). However the band observed for ALDH1A3 was almost twice the
expected size. The size expected was around 900bp and the size seen on the gel
is around 1500bp. Parts cut from the gel were ALDH1A1 (EPD), ALDH3A1 (EPD) and
ALDH2 (Wmin). DNA was gel extracted and purified. DNA
was then run on a gel to verify the extraction process. An overnight digestion
was set up to create the Biobrick overhang parts. Only
the ALDH1A3 digest from previously was kept. The other digests were discarded
at this point. Tuesday Today the restricted parts were cloned to the pSB1C3
backbone and transformed to E. coli. Parts cloned were the ALDH1A1 (EPD),
ALDH2, ALDH1A3 and ALDH3A1. Transformed E.
coli was plated to chloramphenicol plates which were incubated overnight at
370C. More plated and
broth were made up ready for use. The parts required from DTU Denmark and Serrano
are being arranged for delivery to us. The following promoter parts have been mad ready. ALDH1A1 – BBa_K940000 ALDH2 – BBa_K940001 ALDH 1A3 – Bba_K940002 ALDH3A1 – BBa_K940003 Wednesday The linker primers were hydrated ready for use. We are still
waiting for the parts from DTU and Serrano. There have been a number of issues
with delivery but we are hoping to have them sorted and the parts delivered by
Friday. Colony PCR was done to confirm the inserts ligated yesterday. RESULTS: Only some of the colony PCR have worked. This may be due to suboptimal primer
annealing temperatures. Colony PCR was carried out as 10µL volumes an annealing
temperature was 580C using the Pfu polymerase. All colonies were inoculated to LB broth and grown overnight at 370C
in a shaking incubator. Thursday Two samples of each of the LB cultures were selected and
plasmid extracted using the QIAprep kit. PCR was then
done on the extracted plasmid in order to confirm DNA insert. This was done as
we had not managed to get good bands for all the colony PCR’s. The PCR results were variable. Again, this may have been due to
annealing temperatures used in PCR. The samples were analysed on the nanodrop. Nanodrop results were
as follows: But it has now been found that the concentrations we have
are too low to send for sequencing and still have enough for sending to iGEM
parts registry. An attempt to concentrate the samples was made by putting the
samples in a spin vacuum. Disaster!!! I have mixed up the lids! Colonies were
quickly re-picked and inoculated to LB for growth overnight. Friday The parts have arrived from DTU Denmark. PCR has been set up
using Pfu Turbo. DNA template used was as follows: ALDH1A1 (BBa_K940000) extracted
plasmid mCherry (BBa_K678067) DTU terminator (BBa_K678067) DTU hygromycin (BBa_K678049) DTU ALDH2 (BBa_K940000) extracted plasmid Neomycin (BBa_K678067) DTU Biobrick
backbone (pSB1C3) unrestricted RESULTS: Unfortunately we did
not get any bands. Plasmid was visible on the gels which were run. This may
indicate that the plasmid concentration was too high for the PCR reaction. Plasmid
extraction was done using QIAplasmid prep kit. PCR
was done on the extracted plasmid, but again the PCR was not very successful. NEXT STEPS: Plasmid template was diluted 1/1000
and another PCR set up using the Pfu. Hygromycin is the only part which was amplified. Mg2+ was included in the
reaction mix this time. DTU had reported success when they used Mg2+ . We
were able to amplify hygromycin. WEEK 3 Monday The plasmid previously extracted was analysed on the nanodrop. The samples were made ready for sequencing and
for sending to iGEM. Nanodrop results were as follows: ALDH1A1(1): 64.5ng/µL with purity ration 260/280 =
2.13 ALDH1A1(2): 348.5ng/µL with purity ration 260/280
=2.20 ALDH2(1): 59ng/µL with purity ration 260/280 =
1.08 ALDH2(2): 66.8ng/µL with purity ration 260/280 =
1.80 ALDH1A3(2): 166.9ng/µL with purity ration 260/280 =
2.17 ADH1A3(2): 44.5ng/µL with purity ration 260/280 =
2.20 ADH1A3(2): 174.2ng/µL with purity ration 260/280 =
2.20 ADH1A3(2): 201.4ng/µL with purity ration 260/280 =
2.22 PCR using Taq polymerase was set
up. We may be able to amplify the parts using this different polymerase.
Unfortunately such products could not be used for user cloning. Taq PCR was set up according to manufacturer protocol. PCR
was run overnight. Tuesday The overnight
PCR was analysed. Promoter
parts were sent for sequencing. The parts were also packaged and sent to iGEM
registry. ALDH1A1 – BBa_K940000 ALDH2 – BBa_K940001 ALDH 1A3 – Bba_K940002 ALDH3A1 – BBa_K940003 Wednesday CONCLUSION: Unfortunately we have not been able to achieve all that we set out
to do in the lab. In the two and a half weeks which we have had in the lab we were
able to amplify the promoter regions which we had designed and we have sent them to
iGEM registry. These parts are currently being sequenced. Parts produced by the group
ALDH1A1 promoter BBa_K940000
ALDH2 promoter BBa_K940001
ALDH1A3 promoter BBa_K940002
ALDG3A1 promoter BBa_K940003
