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 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 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:

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

The ligated products were transformed.

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 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:

PCR 1: EcoRI + SpeI

PCR 2: EcoRI + SpeI

PCR 3: EcoRI + XbaI

B+C: EcoRI + XbaI

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.

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 streak plating 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):

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