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Team:SDU-Denmark/Tour32
From 2014.igem.org
Lab Journal
Week 25 (16/6 - 22/6)
Monday 16/6
We started our work in the wet lab with a three days lab crash course. We learned basics, as running a PCR, and where we can find everything that is needed for working in the lab. In addition, we talked a lot about the project goals and the way of getting there.
PCR reactions on TetR, both to include constitutive promoter, RBS and terminator, and to include the same elements, but remove LVA-tag, and on GFP generator to include Tet promoter, RBS and terminator.
PCR1: Const.P-RBS-TetR(-LVA)-term.
PCR2: Const.P-RBS-TetR(+LVA)-term.
PCR3: Tetp-RBS-GFP-term.
Tuesday 17/6
In the lab: The PCR reactions from yeasterday didn't work, so they were repeated. This time 50 µL PCR reaction were prepared. Gel purification was performed on the PCR products and the final concentration was measured by nanodrop.
We digested the products by using restriction enzymes, XbaI og SpeI. We detected that the plasmid was only linearized, meaning that only one restriction enzyme/restriction site was functional.
We worked on the basics in the iGEM concept. We discussed the definitions of biobrick, basis part, composite part and devices. We learned the "Standard Assembly Method", it's condition, uses and limitations.
We defined the conditions for our own device and started searching for the necessary parts in the iGEM registry.
Wednesday 18/6
We digested two plasmids, 161 and RFP, each with the enzymes XbaI and EcoRI. Unfortunately it didn’t work as expected, as XbaI did not cut. We made some control experiments to check this and concluded that one of the plasmids might miss the EcoRI restriction site.
The results after running it on a gel shows that somethings wrong with the plasmid, 161, since Xbai doesn't digest it, but digests plasmid, RFP. Since EcoRI digests, we can conclude that there's something wrong with the plasmid's XbaI restriction site.
Several control experiments with the XbaI enzyme was made - another borrowed Xbai enzyme was tested as well. Since the borrowed enzyme worked, the other was tossed out. Conclusion: We're buying a new one!
We recieved lab safety training.
Friday 20/6
Team page and Notebook page added to the wiki.
Two PCR reactions were made to amplify plasmids from the iGEM 2014 kit. The gel, that was run afterwards, showed bands in the right length. The bands were cut out and purified.
Saturday 21/6
All the pipettes were calibrated
A K12 MG1655 strain was plated out on agar, to colonize overnight, in order for us to produce more plasmid later on.
Sunday 22/6
One bacterial colony from the agar plate from yesterday was transferred to fluid LB media to amplify WT bacteria. TSB buffer was made. A transformation was made.
Week 26 (23/6 - 29/6)
Monday 23/6
The transformation from yesterday has not been successful.
New plates with Ampicilin, Kanamycin and Chloramphenicol were made.
Wednesday 25/6
A transformation was made.
Thursday 26/6
The transformation from yesterday has been partly successful. One plate had no colonies. For the transformations that were successful an ON was made.
The cultures should be prepared for storage at -80°C tomorrow.
Friday 27/6
Freezing stocks were made out of the ON from yesterday. The samples were then stored at -80°C.
Saturday 28/6
The non-successful transformation from Wednesday 25/6 was repeated.
Sunday 29/6
The transformation was partially successful. There were some, but very few, visible colonies on the agar plates. Two overnight cultures were prepared from one colony respectively.
Week 27 (30/6 - 6/7)
Monday 30/6
The overnight culture was not red, as expected, which means that something was wrong with the plasmid. Therefore a new overnight culture was prepared, so that a Colony PCR can be performed on the cultures. Furthermore an overnight culture was prepared of the wild type E. coli strain, so that it can be used for transformation tomorrow.
Tuesday 1/7
Today a Colony PCR was performed on the E. coli MG1655 strains with the plasmids: pSB1A3, pSB1C3, pSB1K3 and J04450 (also known as aza). The results showed that the constructs all had approximately the same weight, just below 1500 bp. This corresponds to the theoretical weight of the constructs, which is 1382 bp.
The development of the construct used to produce lacI was commenced. This was done by first running Phusion PCR on lacI (BBa_I732100, Template 8A, Plate 3). The PCR product was then analysed on gel-electrophoresis and purified.
Transformation was performed on the plasmids pSB1C3 and pSB1K3 as the former transformation was unsuccessful. Furthermore transformation was also performed on the parts BBa_R0010 (lacP promoter) and BBa_E0840 (GFP coding seq.). The transformed E. coli were put on agar plates with antibiotic according to their respective resistance. The plates were placed in the heating cabinet and should be examined for colonies tomorrow.
Wednesday 2/7
Because the PCR on lacI from yesterday yielded a low concentration, Phusion PCR was repeated today, where both the PCR product from yesterday and BBa_I732100 from iGEM were used as template. The results showed that the PCR product from yesterday was too short and still yielded a low concentration. While the PCR reaction with BBa_I732100 as template produced a higher concentration of lacI.
The transformation on the parts BBa_R0010 (lacP promoter) and BBa_E0840 (GFP coding seq.) from yesterday was unsuccesful so the experiment was repeated today.
Freezing stocks of E. coli with pSB1C3 and pSB1K3 were stored at -80°C. Each culture were mixed with 50% glycerol
Furthermore fast digest on our 3 PCR products were made with XbaI and SpeI.
PCR 1 and 3 were ligated and PCR 2 and 3 were ligated.
Thursday 3/7
Today the ligated PCR1/3 and PCR2/3 were purified by running them through a gel. The concentration of the purified constructs were measured by nano drop.
In order to create "sticky ends" on PCR1/3 and PCR2/3, the parts were fast digested. Also pSB1C3 was fast digested, so that it could be used as a backbone. The different fast digested parts were gel purified. PCR1/3 and PCR2/3 were then ligated to the backbone (pSB1C3).
Colony PCR was performed on a colony from the transformation from Wednesday 2/7 of the lacP promoter (BBa_R0010) and GFP coding seq. (BBa_E0840). The gel showed bands that were too long, therefore a new colony PCR was performed on a different colony. The results from the new colony PCR were the same as before, with the weight of GFP just above 1000 bp and the weight of lacP promoter just around 500 bp.
Friday 4/7
A miniprep was performed on the ON containing lacP promoter (BBa_R0010) and GFP coding seq. (BBa_E0840), and a freezing stock was made.
Furthermore a PCR was made on the products from the miniprep. Unfortunatly the PCR didn't work, as the gel didn't show any products.
Because the PCR didn't work we found the products from the iGEM kit from 2014 and made a new PCR.
This time the PCR worked and the products were gel purified and the concentrations meassured with NanoDrop.
Saturday 5/7
PCR was preformed on LVA less GFP with tet promoter using primers yellow#8 and yellow#9. The PCR was purified using the gel purification SOP.
A digest was done on PCR products #11 and #12 in an attempt to ligate these. Ligation was done in 30 mins at RT whereupon the ligase was inactivated at 65 °C for 10 mins. Purification of the ligation product was done, diverging from the SOP and resulting in an unusable amount of product.
Sunday 6/7
The lacP promoter (BBa_R0010)and GFP coding seq. (BBa_E0840) have been ligated but the concentration was low and therefore we made PCR on the product.
The PCR product was run on a gel. The band was hard to separate from other bands around the same length (1400bp). We cut out the right length and purified it. Nanodrop was measured.
PCR1 (consisting of template 2:2P) and PCR2 (consisting of template 2:2P) have both been fast digested with SpeI. PCR3 (consisting of template 2:24D) has been fast digested with XbaI. PCR1 and PCR3 were attempted ligated (for 30 min) and so were PCR2 and PCR3. These ligations did not show any bands around the expected length during gel purification. Two new ligations (of the same), with higher concentrations of digested PCR products, are reacting overnight.
Week 28 (7/7 - 13/7)
Monday 7/7
We realized that we have to step back in the process by inserting the different parts we want to ligate into a plasmid before ligating them to each other.
Therefore we started our day in the lab by doing a PCR reaction on PCR1 (consisting of template 2:2P) and PCR2 (consisting of template 2:2P). Unfortunately PCR2 didn't show the right band length when run on gel.
PCR was also done on the lacP promoter (BBa_R0010)and GFP coding seq. (BBa_E0840). The sequences machted the lenght seen on the gel. The band from the gel was cut out and purified.
PCR1, PCR3 and LacP were digested.
Tuesday 8/7
The digested products from yesterday were purified.
Furthermore PCR on PCR 2 was repeated, this time with a gradient spanding from 53°C - 58°C. Our results were good this time. The products were purified and their concentrations were meassured with NanoDrop.
Wednesday 9/7
Tet construct: Plasmid pSB1C3 was concentrated before it was digested with the enzymes Xbai and SpeI. The plasmid was ligated with digested PCR1, PCR2 and PCR3.
Lac Construct:Thursday 10/7
The transformation from yesterday was successful. Colony PCR was performed on all transformations. The result showed that part B (Bba_R0010) and C (Bba_E0840) had been ligated successfully. Unfortunately nothing could be seen on the gel with regard to PCR1, PCR2 and PCR3. Therefore a new colony PCR was performed on different colonies with PCR1, PC2 and PCR3. The gel showed no or no proper bands, so we concluded, that a religation must have happened. Therefore we checked our enzymes by digesting pSB1C3. Here we could conclude that something must be wrong with XbaI, as it does not cut.
An ON was made containing BC construct.Friday 11/7
XbaI was doublechecked and we found out, that the enzyme does not cut when using the green Fast Digest buffer.
We puridied our PCR1 (Pcon_rbs_BBa_C0040(LVAtag_removed)_term) and PCR3 (Ptet_BBa_E0840) and digested these while using the colorless buffer. Furthermore PCR1, PCR2 and PCR3 were amplified by a PCR reaction.
ON containing BC contruct was miniprepped and run on a gel together with C in plasmid. The length of the bands were compared and seemed appropiate. Anyway, to be sure, a PCR was made on the BC construct. This time the gel showed too short bands, which could mean, that the C in plasmid has religated. We conclude that this must have happened because of the enzyme XbaI, that did not cut in green buffer, but was used on this construct before. Therefore C in plasmis was digested again and ligated with B.Saturday 12/7
Ligation of our digested product, PCR 1 (Pcon_rbs_BBa_C0040(LVAtag_removed)_term), PCR2 (Pcon_rbs_BBa_C0040_term) and PCR3 (Ptet_BBa_E0840), into a backbone, pSB1C3. Ligation of B(LacP, BBa_R0010) and C (GFP, BBa_E0840) construct into pSB1C3.
Transformation of PCR 1(Pcon_rbs_BBa_C0040(LVAtag_removed)_term),PCR2 (Pcon_rbs_BBa_C0040_term), PCR3 (Ptet_BBa_E0840) and BC (LacP, BBa_R0010 + GFP, BBa_E0840) into E. coli.
An ON containing plasmid pSB1C3 was made.
Sunday 13/7
The ON containing pSB1C3 from yesterday was miniprepped.
Week 29 (14/7 - 20/7)
Monday 14/7
ON culture containing wild type E. coli from freezing stock was made.
The agar plates with our transformated plasmids from Saturday showed colonies with different colors. Colony PCR was made on the colonies that seemed to have worked and were afterwards ran on gel. The bands had the right length. Furthermore single colony plate streaking was made on the same colonies, that were used for Colony PCR.
B and C in plasmid, and PCR1, PCR2, PCR3 and pSB1C3 were ligated and afterwards transformed.
A PCR was run on PCR1, PCR2 and B (lacI).
Tuesday 15/7
A mini-prep was made. This was not successful, therefore a new ON was made.
Wednesday 16/7
A mini-prep was made on the ON cultures from yesterday (PCR 1, PCR 2, PCR3 and B+C in plasmid). The concentrations were not high enough to be sent to sequencing. The samples were therefore placed in the centrifugal evaporator to increase the concentration.
A new ON culture of these strains is prepared, in case that something goes wrong
Also the ordered parts from iGEM arrived today, and these were plated on agar plates with the appropriate antibiotics and placed in the incubator.
B construct in plasmid was digested with EcoRI and XbaI. The band on the gel was at the right length. The gel was cut out and purified.
New plates containing antibiotics were made.
Thursday 17/7
The plasmids that were prepared yesterday (PCR 1, PCR 2, PCR 3 and B+C) were digested today with:
The products were then run on gel, cut out and purified and furthermore the concentration was meassured with NanoDrop.
PCR1 and PCR2 were ligated with PCR3, and BC and B were ligated with A. All plasmids were transformed.
Furthermore the plasmids with PCR 1, PCR 2, PCR 3 and B+C were prepared to be sent to Eurofins for DNA sequencing.
As mentioned yesterday a ON-culture was prepared. The culture was attempted mini-prepped, but the mini-prep was not successful. Consequently a new ON-culture was prepared today.
The parts from iGEM that were plated yesterday, were today prepared for ON-culture, so that a freezing stock can be made tomorrow.
Friday 18/7
We prepared a freezing stock of the parts we had ordered and received from iGEM, and the remaining sample from the ON culture used for the freezing stocks, was miniprepped and catalogued.
Some of the minipreps, were prepared with a wash buffer not containing any ethanol, and therefore we has miserable results. New ON cultures of those was prepared.Saturday 19/7
The ON cultures of the iGEM parts from yesterday, were miniprepped with fine results.
Colony PCR was performed on the cultures transformed with PCR1+3, PCR2+3, ABC and AB. There were no colonies on one of the plates (AB-part).
Plate streaking was performed on each colony taken for colony PCR.
At first sight the results from the colony PCR seems to suggest that something went wrong during the digestion or ligation, since only re-ligations seems to be present.
Sunday 20/7
As we were not successful in transforming the samples from Saturday 19/7 a new batch was initiated. So PCR1, 2, and 3 and furthermore B and BC were digested, ligated, and transformed.
In accordance with our decision to make the nutrient producing strain taste good transformations of parts; I742111 (RBS+limonene synthase), K118024 (dsx+LIMS1+appY), J23119 (constitutive promoter), and B0015 (double terminator) was commenced. These are going to constitute the construct for producing lemon taste and scent.
Week 30 (21/7 - 27/7)
Monday 21/7
Colony PCR was made on the transformations from yesterday. We concluded that the transformation not have been successful. Therefore we went back and made new digestions.
ON cultures containing J23119, I742111, K118024, Lac promoter and pSB1AT3 were made.
Tuesday 22/7
Minipreps were made on the ON cultures from yesterday.
The digested products from yesterday were ligated and transformed.
Wednesday 23/7
Today we ran colony PCR on the transformations from yesterday. A single colony plate streaking was made for each colony that was used for Colony PCR.
J23119 and K118024 were ligated and J23119 and I742111 were ligated.
Thursday 24/7
We recieved a delta 12 desaturase from Julius Fredens today, it is a Fat2 desaturase originating from C. elegans, we are planing on testing it and we might add it to the iGEM parts registry since it does not seem to be registered.
/Daniel
Friday 25/7
Over night cultures have been made for (started at 3:30 PM):
/Daniel
To check that we have the correct lenghts of our PCR1,2,3 and part A,B,C & BC, we ran a PCR. This did not work, so we repeated the PCR this time with a Tm on 51 degrees. The products with a length on 1000 bp and lower got 15 sec. at step 4, and the products with lengths over 1000 bp got 30 sec.
/Camilla
Saturday 26/7
Miniprep has been made of the ON cultures from 25/7 (these can be used for cloning later on):
PCR reactions of PCR 1, 2 and 3 was made from different templates, these are to be purified tomorrow.
/Daniel
Gradient PCR was ran on part A with lac_LVAR & lac_LVA_F as primers. 2,5 uL template & 31 uL water. Tm graient at 51-70 deg. with 5 samples. PCR program: 1: 98 deg., 2 min. 2: 98 deg., 10 sec. 3: 51-70 deg., 30 sec. 4: 72 deg., 45 sec. 5: GO TO 2 rep 30. 6: 72 deg., 5 min. After running the PCR on a gel, no clear, correct bands showed. This was due to the primers, which had a too high concentration, thus they were diluted.After the dilution, a touch down PCR was made, starting at 66 deg. going 1 deg. lower per cycle for 10 cycles, thus ending at 56 deg. A gel showed a clear band with the correct bands. In order for us to have a high enough concentration of PCR1 & PCR2 for ligation, we ran a PCR with the program: 1:98 deg., 2 min. 2: 98 deg., 10 sec. 3: 50 deg., 30 sec. 4: 72 deg., 2 sec. 5: GO TO 2 rep 30. 6: 72 deg., 5 min.
after running a gel with the PCR products, bands showed with the wrong lenghts that wasn't religation. Thus we concluded that the PCR1 and PCR2 used was incorrect.
Due to this, we ran a PCR on all of our PCR1,2,3 products with verification primers, to check wether they had the correct lengths, which they luckily had.
/Anne, Sarah & Camilla
Sunday 27/7
PCR products made yesterday has been purified, this was PCR 1, 2 and 3 from different templates.
/Daniel
Digest of BBa_I742111, with S+P, BBa_K118024, with X+P, BBa_B0015, with X+P, and pSB1C3 with BBa_J23119, with S+P and FastAP. Followed by ligation of digested BBa_J23119 with digested BBa_I742111, BBa_K118024 and BBa_B0015 respectively.
/Camilla
The Lac construct (ABC) and A-plasmid (LacI) has been digested: PCR A with E+R, BC-plasmid with E+S, and A-plasmid with E+S. FastAP were added for digestion of the backbones. 30 min. digest.
This was followed by purification and measurement of the concentration by nanodrop.
The Tet construct has been digested: PCR1 with E+S, PCR2 with E+S, and PCR3 in plasmid with E+X and FastAP. 30 min. digest, followed by purification and nanodrop.
Following has ligated:
PCR A + pSB1C3
PCR A + BC-plasmid
PCR1 + PCR3
PCR2 + PCR3
In addition a new TSB buffer was made.
/Anne
Week 31 (28/7 - 3/8)
Monday 28/7
The plasmid of BBa_J23119 has been checked to find out, if we took the wrong template. Thus the template: plate 3: 17O has been transformed.
The ligations from yesterday has been transformed as well.
/Victoria
Transformation of PCR1, PCR3 and BC-plasmid has been made earlier. Overnight cultures from these transformations has been miniprepped and saved as freezing stock.
Further, BBa_J23119 has been transformed to save as freezing stock as well.
/Signe
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Tuesday 29/7
Results from Transformations:
PCR A + pSB1C3: Red cultures, high growth - colony PCR shows the expected: Religations!
PCR A + BC-plasmid: White cultures (fine) - colony PCR shows the right length
BBa_J23119: White cultures - overnight culture for miniprep tomorrow has been made.
/Signe
Thursday 31/7
The overnight culture of J23119 has been miniprepped and saved as product.
Since there's no more PCR3 product, an overnight culture of bacterial cells, containing this in plasmid, has been made.
/Camilla
Friday 1/8
The overnight culture of PCR3 has been miniprepped, nanodropped and saved as product. The concentration were too low though - the sample was run in speedy vac.
Digest of PCR1-plasmid with E+S, PCR2-plasmid with E+S, and PCR3-plasmid with E+X.
In addition, freezing stocks has been made, of BBa_I742111, BBa_K118024, BBa_B0015, BBa_J23119 and Fat2 (Δ12 desaturase).
The results from the earlier digests run on a gel showed no PCR3 product, why another gel was run with same PCR3, not digested, to see, if there's no DNA in the sample solution. This showed no bands. An overnight culture has been made from freezing stock of the same PCR3 sample, to investigate, if something's wrong with the freezing stock.
The digested PCR1 and PCR2 had the right lengths and has thus been purified.
/Signe
Saturday 2/8
The sequencing data of PCR3 from specific freezing stock sample, showed that the GFP generator has the wrong direction - wrong ligation links of X-X and S-S (X-S and S-X).
To make a new PCR3 construct, PCR on template: plate 4: 11P, 2014, BBa_E0840, was made, since the template was a pSB1A3 backbone. The product was run on a gel. The bands showed the right length, and the product was thus purified.
pSB1C3 has been digested with S+X and FastAP - reaction for 1 hour. The reaction was run on a gel, and the right band (around 2000 bp) has been purified. In addition, the new PCR3 has been digested as well, with X+S, and purified.
Following ligations has been made:
PCR3-template has been transformed, like the ligations above.
Also, a digestion of the constitutive promoter, J23119, with S+P, has been made. This was run on a gel, and the right band (2000 bp) was purified.
/Daniel
Sunday 3/8
Colony PCR on the transformations from yesterday.
/Martin
Week 32 (4/8 - 10/8)
Monday 4/8
Digest of pSB1C3 with S+X and FastAp, which is needed as backbone for PCR3. This has been run on a gel and the right band was purified.
/Daniel
Overnight cultures of single colony plate streaks from yesterday's colony PCR has been made. Also, a freezing stock of new PCR3 template.
/Victoria
Colony PCR on a single little, beautiful, green flourescent culture from the transformation of PCR3 from 2/8. Three types:
Damnit, nothing!
/Ulrika
BC-plasmid digested with E+S
I74 digested with E+S
BBa_B0015 digested with E+X
PCR3 digested with X+S
Right bands on gel has been purified.
/Martin
Tuesday 5/8
Ligation of PCR3 with pSB1C3, J23/I74 and J23/K11 with B0015-plasmid.
/Signe
Transformations of ligations above.
/Martin
Wednesday 6/8
Colony PCR on transformations from yesterday. This showed the expected lengths.
/Daniel and Victoria
Thursday 7/8
Overnight cultures from single colony plate streaks of the colonies used for colony PCR yesterday.
Every earlier product containing PCR3 has been thrown out, since the new PCR3 in plasmid has the right length and direction, we won't risc mixing it up with old products with wrong directions.
/Victoria
The new PCR3 has been miniprepped and digested with E+X and FastAP. Meanwhile, a new colony PCR on the transformation of PCR3 from 5/8 was made again, just to make extra sure that the PCR3 constuct was right.
/Martin
Ligation of PCR1 + PCR3 and PCR2 + PCR3.
/Ulrika
Ligations above has been transformed.
/Martin
Friday 8/8
The transformations were shit. Nothing worked. Either we have used the wrong strain, or something's wrong with the antibiotica plates.
New primer stocks has been made.
JIB and JKB has been miniprepped.
/Martin
Digest of C-plasmid with X+P and digest of B-plasmid with S+P and FastAP. Only the B digest was purified, and digestion og C was made again - still didn't work.
/Camilla
Overnight culture of Fat2, which will be miniprepped tomorrow.
A new PCR3 product has been made with new primers.
/Martin
Transformation of the same ligations from yesterday.
In addition, a new PCR on PCR3 template has been made with the new primers, and on A template with the new primers. PCR3 was purified from gel - PCR A didn't work.
Digest of PCR3 with X+P (since the new primers contain a P-site), PCR1 with S+P, and PCR2 with S+P. These three digests were pusified.
Ligation of PCR1+3 and PCR2+3 - transformation of these ligations.
/Ulrika
Saturday 9/8
Plating of WT on Cm plate from 8/8 didn't show any colonies, so there's probably nothing wrong with the plates.
Freezing stock of the new PCR3, JIB and JKB has been made.
Fat2 has been miniprepped and the concentration was measured by nanodropping.
Transformations from yesterday showed neon green colonies on all the plates, also the control plates. Colony PCR has been made - this showed wrong bands, and the transformations thus didn't work.
/Signe
PCR reaction on A, C and Fat2 has been made - only Fat2 showed product on a gel and was purified.
/Daniel
Digest of C-plasmid with X+P. Was run on gel and purified.
Ligation of C-part in B-plasmid.
In addition, an overnight culture of PCR3 template has been made (GFP generator).
/Camilla
New PCR, once again, on A with new primers - once again, failed!
/Daniel
Sunday 10/8
Overnight culture on pSB1C3 and GFP generator.
Digest of Fat2 with X+P, and pSB1C3 with X+P and FastAP.
/Daniel
New colony PCR on transformation of PCR1+3 and PCR2+3. This showed the right lengths in some of the colonies.
/Jens Jakob
Gradient PCR on A with diluted primers - didn't work.
Touch down PCR on A - didn't work.
/Camilla
Transformation of CB-plasmid and Fat2 in pSB1C3.
/Martin
Week 33 (11/8 - 17/8)
Monday 11/8
Miniprep of PCR1/3 and PCR2/3 overnight cultures.
/Camilla
Freezing stock of PCR1/3 and PCR2/3.
/Jens Jakob
Tuesday 12/8
Colony PCR on transformations from 10/8 - both Fat2 and BC construct showed the right lengths.
/Signe
Wednesday 13/8
Colony PCR on single colony plate streak from transformation of BC construct. The colonies from the single colony plate streak were neon yellow. Some of the bands had the right length, and overnight cultures has been made from these, so feezing stock can be made and miniprep can be sequenced.
New solutions of specifik primers from stock has been made, and PCR reactions on the same A template, with old and new primers, has been made to test the new primers.
- THE NEW PRIMERS WORK! And the old primers has been tossed out - accidently the gel was tossed out during the exitement as well.
Digest of Fat2 with X+P
Digest of pSB1C3 with X+P and FastAP
- Fat2 is too long and has to be digested again.
- The length of pSB1C3 matches, and the product has been purified from the gel.
/Signe
Once again, another PCR reaction on A - didn't work!
PCR reaction on Fat2 - product has been gel pruified, digested with X+P, and purified.
Overnight culture of single colony plate streak from transformation of BC has been made.
/Martin
PCR reaction on three different templates of A with new primers - every reaction worked and has been purified from the gel.
In addition to this, template plasmid of A has been transformed to amplify this.
Ligation of digested Fat2 with pSB1C3 - this ligation has further been transformed.
/Ulrika
Thursday 14/8
Overnight culture with BC has been miniprepped and freezing stock has been made.
Digest of A with E+S and BC-plasmid with E+X and FastAP. This has been run on a gel and purified from this.
Colony PCR on transformation of Fat2 - the bands has the right length on the gel.
/Signe
BC has been sent for sequencing.
We have recieved USER primers.
Overnight cultures of Top10 with pET::PfuX7 plasmid and BL21(DE3)pLysS has been made, for the protein purification of USER polymerase.
/Daniel
Ligation of A with BC - this ligation has been transformed.
/Ulrika
Friday 15/8
Freezing stock of Top10 with pET::PfuX7 plasmid and BL21(DE3)pLysS has been made.
/Daniel
Overnight culture on the single colony plate streak from the transformation of Fat2 has been made.
ON of A-plasmid has been miniprepped and made freezing stock of.
Colony PCR on ABC transformations - some had yellow colonies, but many had white. The gel showed that many of the colonies contained the right length (2508 bp).
/Signe
Saturday 16/8
ON culture of Fat2 fra 15/8 has been miniprepped and made freezing stock of. On monday, these has to be sent for sequencing.
/Signe
PCR reaction on PCR3 - didn't work!
ON on the ABC single colony plate streak from 16/8 has been made.
/Camilla
Sunday 17/8
Freezing stock of the ON on ABC from yesterday has been made.
The rest of the ON has been miniprepped.
ON of Top10 BL21 with pET::pfuX7 has been made.
/Daniel
ON of BL21 (DE3) plysS, Cm, and Top10 with pET::pfuX7, Amp, has been made.
pSB1C3 containing bacterial cells has been plated Amp/Cm, to investigate, if Amp works.
/Jens Jakob
New PCR reaction on PCR3 template. The product has been purified from gel and digested with X+P. This has been ligated with PCR1 and PCR2 respectively, both as backbone, digested with S+P. (This PCR2 has been shown to have the right sequence).
/Ulrika
Week 34 (18/8 - 24/8)
Monday 18/8
Transformation of the ligations from yesterday.
Two transformation were made, due to stupid mistakes.
In addition pSB1C3, digested with X+P, has been ligated with PCR3, digested with X+P.
This ligation has been transformed.
/Ulrika
Tuesday 19/8
Colony PCR and single colony plate streaking on and from the transformations from yesterday - all transformations from yesterday showed to be successful!
/Jens Jakob
Wednesday 20/8
A SDS buffer has been made.
/Signe
Digest of BC-plasmid with E+X - was purified from gel.
/Camilla
Ligation of A: E+S, with BC-plasmid: E+X, newly digested by Camilla because of mutations in the old BC construct.
This ligation has been transformed.
/Ulrika
ON of PCR1/3, PCR2/3 and PCR3 has been made - taken from single colony plate streaks.
/Jens Jakob
Thursday 21/8
Freezing stock on ON of PCR1/3, PCR2/3 and PCR3-plasmid.
These ONs has also been miniprepped.
Colony PCR on ABC - showed a little success.
Digest of A with E+S for 20 min. - has been purified.
Ligation of this with BC.
/Camilla
Friday 22/8
A forgotten control of the ligation from yesterday has been made.
- Trasnformation of the ABC ligations.
/Camilla
Saturday 23/8
BC from two different templates has been digested again, with X+E - only one of the digestions worked. This has been purified from gel and ligated with A: E+S.
/Ulrika
New Cm plates has been made and the ligation above has been transformed.
/Ulrika
Sunday 24/8
Colony PCR on transformation of ABC - several of the colonies showed to contain the right length (2660 bp).
/Jens Jakob
Week 35 (25/8 - 31/8)
Monday 25/8
New colony PCR on ABC single colony plate streaks from yesterday - one colomy was correct, and ON has been made.
/Martin
Tuesday 26/8
From the ON of ABC, freezing stock has been made and miniprep has been sent to sequencing.
/Martin
Wednesday 27/8(
Preparation for iGEM meet-up in London.
Thursday 28/8
Different cultures (8 ON) has been made ready for FACS:
2 x WT, 2 x PCR3, 2 x PCR1/3, and 2 x PCR 2/3.
ON of JKB has been made for miniprep tomorrow.
/Martin
Friday 29/8
ONs of JIB, JKB and J23119 has been miniprepped.
Digest of JIB and JKB with X+P, and of J23119 with S+P and FastAP - all was purified from gel.
Ligation of JIB+J23119 and JKB+J23119 has been made.
We got Bacillus subtilis on two plates. An ON of this has been made for freezing stock tomorrow.
PCR reaction on B. subtilis has been made - didn't work.
/Camilla
Saturday 30/8
Freezing stock of B. subtilis has been made.
/Martin
Sunday 31/8
Off to London!
Week 36 (1/9 - 7/9)
Monday 1/9
iGEM meet-up in London.
Tuesday 2/9
iGEM meet-up in London.
Wednesday 3/9
Back to Denmark!
Thursday 4/9
Digest of JKB, since there was no more left - purified from gel.
New colony PCR on B. subtilis - didn't work.
Friday 5/9
Miniprep of ONs of BBa_K1027001 (Δ9 desaturase), BBa_K1027002 (Δ12 desaturase), BC construct and Fat2, to be used for USER cloning.
/Anne
Saturday 6/9
ON on ABC, sequenced, maybe correct, to use for A as backbone for USER cloning.
/Ulrika
Colony PCR with phusion polymerase on B. subtilis - didn't work!
/Anne
Sunday 7/9
Miniprep of ON of ABC construct - speedy vaced for higher concentration.
USER PCR on A-backbone, B and Δ9 desaturase - didn't work!
Transformation of ligations from 6/8(?), of JIB and JKB.
/Jens Jakob
Week 37 (8/9 - 14/9)
Monday 8/9
PCR on B. subtilis - didn't work! But we now know why: This strain of B. subtilis doesn't contain the gene we were looking for (which was Δ15 desaturase).
/Ulrika
New USER PCR on A-backbone, B and Δ9 desaturase, under new conditions - didn't work! There were no bands on the gel, only a wierd-looking one for the PCR on Δ9 desaturase, which was 1000 bp too small.
ON on transformations of JIB + JKB from yesterday.
/Jens Jakob
Tuesday 9/9
Miniprep of ON of JIB and JKB from yesterday.
/Victoria
PCR on BBa_1732100 (LacI template) for new A product, since the sequence data from the old one wasn't correct. This was purified from the gel.
/Ulrika
Wednesday 10/9
Digest of PCR2 and PCR3 with S+P and X+P, respectively - prified from gel.
/Camilla
Digest of A from yesterday, with E+P, and pSB1C3 with E+P and FastAP - This was purified (pSB1C3 from gel).
Ligation of these two products, followed by transformation.
Furthermore, A has been digested with E+S, and BC-plasmid with E+X and FastAP - this was purified (BC from gel).
Ligation of A+BC and PCR2+PCR3, followed by transformation.
/Ulrika
USER gradient PCR on A-backbone, B, Δ9 desaturase, Δ12 desaturase and Fat2 (Δ12 desaturase). This didn't show any bands on the gel. Damnit!
/Anne
Thursday 11/9
Colony PCR on single colony plate streaks from the transformations of PCR2/3, A-plasmid and ABC. No bands showed on gel - something is wrong with the conlony PCR?
/Anne and Jens Jakob
Saturday 13/9
USER PCR on Fat2, Δ12, Δ9 and B. The reaction run as a gradient PCR - this didn't work. Only a mysterious and weak band at around 2000 bp shows for all the reactions.
Colony PCR on an empty pSB1C3 vector, ABC, A and PCR2/3 - from the single colony plate streaks from 11/9 - this didn't show any bands on the gel.
/Jens Jakob
Digest of PCR1 and -2 with S+P and self-designed protein with X+P - PCR1 and -2 was purified from gel and the protein didn't show any bands.
/Martin
The protein was digested again, with a bigger amount of DNA - the product was purified from gel and ligated with PCR1 and -2, respectively.
A new colony PCR on the transformations of the empty vector, ABC, A-plasmid and PCR2/3 was made - this didn't show any bands. Again!
/Camilla
Sunday 14/9
A new attempt on USER PCR on Δ9, once again with a gradient - and once again, it didn't work. (The results are like earlier, with a mysterious band at 2000 bp).
/Jens Jakob
Since colony PCR hasn't been working lately, 5 different MyTaq mixes was tested. They all worked, some better than others(?) At the same time, it showed that PCR2/3 had the right length - ON of this has been made.
/Camilla
Week 38 (15/9 - 21/9)
Monday 15/9
Ligation of A in plasmid (A with E+P and pSB1C3 with E+P) and ABC (A with E+S and BC with E+X) - the same as on 10/9. These ligations has been transformed, as well as PCR1-protein, PCR2-protein and J23119-(new lemon flavor template).
Furthermore, WT has been plated on Cm plates, since ABC and A transformations didn't work over the weekend - we're checking WT!
/Ulrika
PCR on A - didn't work!
/Anne
Another attempt on USER PCR on Δ9 - didn't work!
Transformation of the ligations from today.
/Ulrika
Tuesday 16/9
ON of PCR2/3 was contaminated, so a new one was made.
Colony PCR on ABC, A-plasmid and J23119 transformations - all had the correct length on the gel.
/Camilla
Thursday 18/9
PCR reaction on three different A templates. Both with old and new solution of primers, and old and new dNTP solutions. Two of the products showed the right length on gel and has been saved as products.
/Anne
Digest og S with E+S, and digest of J23 with P+S, which has been purified and saved as products.
/Daniel
Friday 19/9
Overnigth culture of bacteria containing the protein sequence has been diluted to an OD ~0.2 and induced with 1000x inducer. This has been standing in 4 hours.
PCR on IB and KB (lemon flavour) with verification primers. This showed the right lengths on a gel.
/Camilla
Digest of two different PCR A with E+S - this has been purified and saved as products.
Ligation of ABC constructs with three different A products: ABC1, ABC2 and ABC3, respectively.
These ligations has been transformed,
/Ulrika
Saturday 20/9
ON on BC product has been made, and freezing stock of ON of PCR2/3.
/Jens Jakob
Colony PCR on ABC transformations and an empty pSB1C3 vector. ABC2 and -3, and the empty vector showed the right bands on a gel.
/Anne
Sunday 21/9
PCR1 and -2 has been digested with S+P and FastAP. The protein ON has been miniprepped and digested with X+P.
J23 has been digested with S+P and FastAP. IB and KB has been digested with X+P.
A has been digested with E+S. BC has been digested with E+X and FastAP.
The protein and IB didn't show usable bands on a gel, and has been digested again.
ON of IB has been made.
Ligations:
This has been transformed, as well as the miniprep of A and the protein.
ON of ABC1, -2, and -3, and an empty vector.
/Martin
Week 39 (22/9 - 28/9)
Monday 22/9
Miniprep of IB and pSB1C3 has been digested, both with X+P. Reaction for 30 min. - this has been purified from gel and saved as products.
Ligations:
Furthermore, an ON of WT has been made.
/Jens Jakob
Tuesday 23/9
ON of odor free E. coli strain for IMS.
ON of protein for maxiprep.
/Daniel
Wednesday 24/9
The Tet promoter, template BBa_R0040, has been transformed.
/Ulrika
Maxiprep of the protein sequence has been made, and this was speedy vaced for higher concentration.
/Victoria
Colony PCR with phusion polymerase on Synechocystis sp1608 has been made, to take out the Δ15 gene - this didn't work!
/Camilla
Freezing stock of the ABC Martin transformed, and ON of another ABC.
First ABC has been miniprepped.
/Anne
Thursday 25/9
Miniprep of ONs of ABC, JIB and JKB - these has been sent for sequencing.
Digest of protein with E+P and pSB1C3 with E+P - this was purified from gel.
/Camilla
Friday 26/9
Ligation of the protein with backbone.
/Jens Jakob
Transformation of the ligation above.
/Anne
Transformation of Tet promoter has been repeated.
/Jens Jakob
Saturday 27/9
Transformations of the protein look a bit suspicious! There's no colonies on the control plate, which is good. But since the colonies on the other plates are all red, we expect it to be religations.
Colony PCR on the transformations shows religations as well.
Colony PCR on transformations of Tet, on the other hand, shows the right lengths.
/Anne
Digest of protein and pSB1C3, both with E+P and X+P (one of each). This has been purified from gel.
Protein, E+P, has been ligated with pSB1C3, E+P, has been ligated, and protein, X+P, has been ligated with pSB1C3, X+P. This has been transformed as well.
/Jens Jakob
Sunday 28/9
Colony PCR on transformations from yesterday showed all successful ligations and transformations.
ON of Tet promoter has been miniprepped and digested with S+P and FastAP. The protein has been digested with X+P. This has been ligated.
IB and KB has both been digested with X+P and ligated with the digested Tet promoter.
New PCR reaction on pBad failed.
The ligations has been trasnformed.
/Jens Jakob
Week 40 (29/9 - 5/10)
Monday 29/9
ON of the single colony plate streaks from the colony PCRs from yesterday has been made to make freezing stock tomorrow - two different protein-pSB1C3.
Colony PCR on the transformations from yesterday has been made. Some of the results looked a bit strange, but ON has been made of Tetp-protein, Tetp-IB and Tet-KB.
/Jens Jakob
Tuesday 30/9
PCR on the protein with primers to make basic part, and on TetR(no LVA) to make basic part. This showed the wrong bands, but the primers seemed to anneal. Very wierd!
A new PCR reaction has been prepared to run overnight.
/Ulrika
Wednesday 1/10
The PCR from yesterday agai showed the wrong bands on a gel. But we found out why: The stock primers used wasn't dissolved! A new gradient PCR reaction has been made and every temperature showed product of both protein and TetR(no LVA) - this has been purified from the gel.
/Daniel and Jens Jakob
The protein (basic part) has been digested with E+P, the TetR(no LVA) (basic part) has been digested with E+P, and pSB1C3 has been digested with E+P and FastAP. This was purified.
Protein and backbone has been ligated.
TetR(no LVA) and backbone has been ligated.
The ligations above has been transformed, as well as PCR3, PCR1/3, PCR2/3, ABC2 and A-plasmid.
/Ulrika
Thursday 2/10
Miniprep of PCR1 and ABC.
/Camilla
Experiments with FACS was done - on the expression of GFP, regulated by Tet repressible promoter, with TetR with and without LVA-tag. But unfortunately, the spectrophotometer were acting a bit strange, and the OD became too high, before doxycycline was added.
/Jens Jakob and Signe
Ligation of protein+pSB1C3 and TetR+pSB1C3 was made.
In addition, new Cm stock was made and new LA plates with Cm.
/Jens Jakob
A new PCR on pBad was made - this didn't work!
/Signe
Friday 3/10
ONs of Fat2 and PCR1/3 was miniprepped and sent for sequencing.
/Camilla
Transformation of PCR3, PCR1/3, PCR2/3, A and ABC2 was made.
/Jens Jakob
Saturday 4/10
Gradient PCR on pBad - showed no bands on a gel.
/Jens Jakob
A new gradient PCR reaction was made, both with BC- and HF buffer. The reactions with BC buffer showed weak bands at all temperatures, which was purified from gel.
/Signe
Colony PCR on transformations from yesterday showed some success - ON was made on ABC2 and A-plasmid.
Something went wrong with the PCR maschine in wihch the colony PCR reactions for TetR and the protein was run. The product was all dried out when this was discovered. A new colony PCR with this was made, and also with new colonies from transformation of PCR1/3 and PCR2/3, since the last result of these were pretty wierd.
The new colony PCR showed succes for all 4 types.
/Jens Jakob
Digest of pBad with E+S, and IB and KB with E+X - this was purified. Both IB and KB was ligated with pBad (but every digest had very low concentration).
/Anne
Sunday 5/10
ON of TetR, protein PCR1/3 and PCR2/3, single colony plate streaks from yesterday, has been made.
/Daniel
Single colony plate streaks were made yesterday. Another colony PCR reaction was run on these, since it contained different cultures (white and green) - with VF2 and specifik reverse primer, and VR and specifik forward primer. The results showed that all colonies contained GFP, but not all contained TetR. ONs were made on 6 colonies, with and without doxycycline.
Result: The colonies that were green before, was green in the ONs as well - with and without doxycycline.
/Jens Jakob
Week 41 (6/10 - 12/10)
Monday 6/10
Western blotts on protein expression were made.
/Victoria
Thursday 9/10
Colony PCR on single colony plate streaks of PCR3, PCR1/3 and PCR2/3, plated from freezing stock, which has been sequenced. Two reactions were made of each: One with specifik GFP forward primer and VR, and one with specifik TetR reverse primer and VF2. The gel showed that all contained GFP and PCR1/3 and PCR2/3 contained TetR, which was expected.
/Jens Jakob
Saturday 11/10
Transformation of TIB, TKB and Tetp-protein in odor free bacterial cells. The cells were transformed at OD600 = 0,3 with 0,5 µl of plasmid.
/Jens Jakob
Sunday 12/10
Colony PCR were performed on 4 colonies from each plate, and they were plated out on new Cm-plates. The colony PCR ran with VF2 and VR primers at 49 deg with 2.30 min elongation time.
Something went wrong with the colony PCR and no bands were observed on the gel. Not even the usual primer dimers in the bottom. This indicates that either the primers, polymerase or the Cm-plates haven't been working.
The primers were investigated by running a PCR on a mini-prepped plasmid with MyTaq polymerase. Bands were observed on the gel, which confirmes that neither the primers or the polymerase was faulty.
A transformation on TIB, TKB and Tet-Prot into the odor free strain, YYC912, were performed once again.
TIB: 0,25 µl and 0,50 µl
TKB: 0,50 µl and 1,00 µl
T-Prot: 0,50 µl and 1,00 µl
Furthermore PCR 1/3 and PCR 2/3 were transformed again, as earlier this week, and it was sent to be sequenced. They were transformed into competent WT cells. They were all plated on a different batch of plates, than what was used last.
/Jens Jakob
