Team:SDU-Denmark/Tour51

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.

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 deviced. 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 plasmid, 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 E restriction site.

We also 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 ran 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 colony PCR (iGEM2014_SOP0021) 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 (igem2014_SOP010) 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.

-JJ

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 consisted of template 2:2P, primer yellow#5 and yellow#6.

PCR 2 consisted of template from previous made PCR2 and primers yellow#5 and yellow#7.

PCR 3 consisted of template 2:24D, primer yellow#8 and yellow#9.

PCR 1 and 3 were ligated and PCR 2 and 3 were ligated. they are stored at 16°C overnight. - Anne

Thursday 3/7

Today the ligated PCR1/3 and PCR2/3 was purified by running them through a gel. The concentration of the purified constructs were measured by nano drop:

PCR1/3 = 2.8 ng/µL

PCR2/3 = 2.9 ng/µL

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 was then ligated to the backbone (pSB1C3) and PCR2/3 was ligated to the backbone (pSB1C3).

Colony PCR was performed on a colony from the yesterday transformation of the lacP promoter (BBa_R0010) and GFP coding seq. (BBa_E0840). Because the results showed that the sequences consisted of more base pairs than they theoretically should, 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.

- Jens Jakob

Friday 4/7

Today we startet with miniprep on our e. Coli containing lacP promoter (BBa_R0010) and GFP coding seq. (BBa_E0840)

A freezing stock was then made from the e. Coli with lacP promotor and with the GFP coding seq.

Further more there was made PCR on the products from the miniprep. Unfortunatly the PCR didn't work because 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 PCR again.

This time the PCR worked and the products were gel purified and we then measured the concentration with nanodrop:

lacP promoter (BBa_R0010) = 20,1 ng/µL

GFP coding seq. (BBa_E0840) = 27,9 ng/µL

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.

- Daniel

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 gel. The band was hard to separate from other bands around the same length (1400bp). We cut the right length out and purified it. Nanodrop was measured to 53,3 ng/µL

PCR1 (consisting of template 2:2P, primers yellow#5 and yellow#6) and PCR2 (consisting of template 2:2P and primers yellow#5 and yellow#7) has both been cut with SpeI. PCR3 (consisting of template 2:24D and primers yellow#8 and yellow#9) has been cut with Xbai. PCR1 and PCR3 was attempted ligated (for 30 min) and so was PCR2 and PCR3. These ligations did not show any bands around the expected length (1700 bp) during gel purification. Two new ligations (of the same), with higher concentrations of digested PCR products, are reacting overnight.

- Anne and Ulrika

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.

We then started our day in the lab by doing a PCR reaction on PCR1 (consisting of template 2:2P, primers yellow#5 and yellow#6) and PCR2 (consisting of template 2:2P and primers yellow#5 and yellow#7). Unfortunately PCR2 didn't show the right sequence when run on gel. This means we will have to repeat our PCR 2 from before to se if it works in second round.

PCR was also done on the lacP promoter (BBa_R0010)and GFP coding seq. (BBa_E0840). The sequences machted the the lenght seen in the gel. The band from the gel was cut out and purified.

- Anne

Tuesday 8/7

Yesterday the following products were digested:

PCR 1, Pcon_rbs_BBa_C0040(LVAtag_removed)_term

PCR 3, Ptet_BBa_E0840

LacP, BBa_R0010

These products were purified today.

Furthermore PCR on PCR 2 (Pcon_rbs_BBa_C0040_term) 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 varied from 10 ng/µL - 11,7 ng/µL.

- Anne

Wednesday 9/7

Tet construct: The plasmid pSB1C3 was concentrated before it was digested with the enzymes Xbai and SpeI. The plasmid was ligated with the following:

The digested PCR1, Pcon_rbs_BBa_C0040(LVAtag_removed)_term, from yesterday was ligated into plasmid

PCR2, Pcon_rbs_BBa_C0040_term, was digested with XbaI and SpeI to begin with and then ligated into plasmid

The digested PCR3, Ptet_BBa_E0840, from yesterday was ligated into plasmid

Lac Construct:

Pcon_rbs_LacI(withoutLVA)_term, BBa_C0012, was digested with EcoRI and SpeI

GFP, BBa_E0840 in plasmid has beed ligated

LacP, BBa_R0010, was ligated with our C part in plasmid

Transformation is made and we will have the results tomorrow.

Thursday 10/7

The transformation from yesterday was successful. To examine if the digestion and ligation were a success, colony PCR was performed on all of the cultures. Four colonies were tested from each agar plate. 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. Therefor a new colony PCR was performed on the colonies with PCR1, PC2 and PCR3, but with VF2 and VR primers.

Friday 11/7

Today we realized that XbaI didn't work well when we used green buffer for fast digest of our products. This leaded to an experiment where we compared digestion with green buffer and a colorless buffer, which we ran on gel afterwards. We saw that the green buffer didn't work optimally because the gel showed smear and there was very little of the disired product in general. We puridied our PCR products 1 (Pcon_rbs_BBa_C0040(LVAtag_removed)_term) and 3 (Ptet_BBa_E0840) and digested these with the colorless buffer this time.

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) konstruct into pSB1C3. Transformation on 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.

Sunday 13/7

We made miniprep to get more plasmid pSB1C3, which can be used in experiments.

Week 29 (14/7 - 20/7)

Monday 14/7

Over night culture was made on our wild type E. coli from freezing stock. The agar plates with our transformated plasmids from Saturday showed colonies with different colors. Colony PCR on the colonies that seemed to have worked was made and run on gel. The lenghts from colony PCR products had the right lengths. There was also made plate streaking from the colonies used for the colony PCR.

PCR was made on PCR 1 (Pcon_rbs_BBa_C0040(LVAtag_removed)_term) (green 20), PCR2 (Pcon_rbs_BBa_C0040_term) (green 22), PCR3 (Ptet_BBa_E0840) (green 21) & lacI (part B, blue 7) using the protocol iGEM2014_SOP0010_v01_Phusion PCR.

Added 1μL template to each reaction -> 13,4 μL water.

PCR program:

1. 98˚C 2 min.
2. 98 ˚C 10 sec.
3. 53 ˚C 30 sec.
4. 72 ˚C 15 sec.
5. GOTO 2REP 30
6. 72 ˚C 10 min.
7. HOLD 4 ˚C

PCR didn’t work on PCR2, PCR3 and lacI (part B, blue 7)

- Camilla J

Tuesday 15/7

Today we made colony PCR from the colonies we plate streaked yesterday. The PCR was repeated from yesterday to double check if our results were right. We had good results again which means we can make freezing stocks on our transformed E. coli.

Wednesday 16/7

Today we started by doing miniprep of the ON cultures from yesterday (PCR 1, PCR 2, PCR3 and B+C in plasmid). Though the concentration was not high enough to be sent in to sequencing. The samples were therefor 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.

_MVM

Thursday 17/7

The plasmids that were prepared yesterday (PCR 1, PCR 2, PCR 3 and B+C) were digested today with:

PCR 1: EcoRI + SpeI

PCR 2: EcoRI + SpeI

PCR 3: EcoRI + XbaI

B+C: EcoRI + XbaI

The products were then run on gel and gel purified. The products were dissolved in 20 µL ultra pure water. The following concentrations were obtained:

PCR 1: 37.3 ng/µL

PCR 2: 8.0 ng/µL

PCR 3: 15.1 ng/µL

B+C: 16.4 ng/µL

The following digested parts were then ligated:

PCR 1 + PCR 3

PCR 2 + PCR 3

A + (B+C)

A + B

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 yesterday. The culture was attempted mini-prepped, but the mini-prep was unsuccessful. 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.

- Jens Jakob

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 plate 5.10 (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 th 19th of June a new batch was initiated. That is PCR1, 2, and 3 together with B and BC was 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.

/Daniel

Monday 21/7

Text

/Name

Tuesday 22/7

Minipreps were made on (yields were measured in nanodrop):

Blue#51: J23119 (104,7 ng/μL)

Blue#52: I742111 (58,8 ng/μL)

Blue#53: K118024 (71,7 ng/μL)

Blue#54: Lac promoter (BBa_R0010) (40 ng/μL)

Blue#55: pSB1AT3 (84,9 ng/μL)

/Daniel

Wednesday 23/7

Today we ran colony PCR on transformations:

PCR 1+3

PCR 2+3

A+B

A+BC

B0015 terminator

Also J23119 and K118024 were ligated and J23119 and I742111 were also ligated.

/Daniel

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):

PCR 1

PCR 2

PCR 3

B

C

BC

/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 1

PCR 2

PCR 3

B

C

BC

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