Team:KU Leuven/Journal/FFL/wetlab
From 2013.igem.org
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
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Weekx
Tekst
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Week4: Getting started
This week we started working on the proof of concept of the feed forward loop in the lab. First of all we selected the necessary bricks and took the plasmids out of the iGEM distribution kit plates. We transformed them into chemically competent inoue cells (which were made before and stored at -80°C) according to the ‘transformation kit protocol’.
Following bricks were transformed: C0076, K823016, C0077, R0080, P0340, R0078, K082003, C0080 and R0011.
All of them were plated out in the presence of chloramphenicol, except for R0011 which is resistant to ampicilline. We made 2 plates for every brick, one with 100 µl and one with 1000 µl LB. The bacteria were grown overnight at 37°C.
The next day we checked our plates and all of them had colonies except for the plates of R0011. We picked one colony of every plate and put the bacteria into tubes with 4 ml of LB medium and 4 µL chloramfenicol to incubate overnight in a shaking incubator at 37°C. Then we decided to transform the failed brick (R0011) and an extra brick (C0060) by electroporation because the efficiency of this method should be higher. We plated them out and let them grow overnight at 37°C.
The plates with brick C0060 had nicely grown colonies and we put them at 4°C for usage next week. The plates with brick R0011 didn't show colonies. Afterwards we noticed that we used the wrong antibiotic resistance. We took the tubes with the successful bricks out of the shaking incubator and we used 400 µl of the LB with bacteria together with 400 µl of glycerol to make a stock in the -80°C.
The remaining 3.6 ml was used for a plasmid extraction using the home-made protocol. After the extraction we used the NanoDrop to see if we had a good amount of DNA and no interference of proteins or salts. The values are shown below:ng/µl 260/280 260/230 C0076 A 94.6 1.34 0.55 B 10.9 1.37 0.41 K823016 A 12.5 1.29 0.43 B 8.7 1.34 0.38 C0077 A 4.1 1.91 0.48 B 2.9 1.70 0.46 R0080 A 18.1 2.02 0.71 B 4.9 1.23 0.32 P0340 A 73.3 1.83 0.99 B 155.8 1.92 1.52 R0078 111.6 1.73 1.07 K082003 A 127.3 1.89 1.55 B 197.1 2.11 1.29 C0080 A 200.6 1.81 1.24 B 7.2 1.92 1.04 -
Week 5: Digestions, digestions and some more digestions.
This week, we decide to do our first digestions. We digested the GFP brick (K082003) with SpeI and EcoRI and the brick with the double terminator (B0015) with XbaI and EcoRI. We put the digested GFP brick on gel in three lanes. After running the gel, we cut the gel in two: one part containing the ladder and one lane with GFP, the other part containing two lanes with GFP. We only stained the part of the gel containing the ladder, this to avoid mutations in the insert of the two other lines. This lanes were used for gel extraction (and ligation afterwards), the lane next to the ladder was used to determine where the insert was located on the gel. After visualisation we marked where our digest was and cut out the part at the same height in the other gel. Afterwards the second gel was stained to check if the bands were cut out correctly. Then we used a kit for gel extraction of the GFP fragment.
digested GFP brick (vector+insert)
After we dephosphorylated the digested vector with terminator and did a pcr purification with a kit, we ligated the GFP insert in this vector according to the ‘Digestion and ligation protocol’. We transformed the product by electroporation. The day after no colonies had appeared, which was expected because the electroporation machine indicated that there was no DNA in the sample (which is of course not possible). So we did the transformation again, but this time with chemically competent inoue cells according to the ‘transformation kit protocol’. Colonies appeared and we inoculated 2 of them in 4 ml LB with chloramphenicol.
We also inoculated some bricks that were stored at -80°C for usage in the next few weeks, namely B0015, B0034 and K608002. We did plasmid extraction of these and of the cells with GFP + terminator next day. We used the NanoDrop to measure the concentrations shown below. We digested 10 µl of the construct with GFP and terminator with EcoRI and PstI to confirm if the right insert is present. This was indeed the case, as you can see on the gel.
ng/µl 260/280 260/230 K082003+B0015 A 141.1 2.05 2.18 B 82.6 1.63 1.65 B0015 A 93.1 2.12 1.75 B 51.6 1.72 1.49 C 106.6 2.08 1.78 B0034 A 317.2 1.92 2.01 B 300.7 1.94 2.12 K608002 A 110.9 1.87 1.32 B 145.9 1.89 1.51 Because we didn’t get high plasmid concentrations for all of the bricks we transformed in week 4, we inoculated a colony from the plates containing K823016, C0060, R0080, C0076 and C0077 in 4ml LB, in the presence of chloramphenicol. The bacteria were grown overnight in the shaking incubator at 37°C. The day after we extracted the plasmids, but this time we used a kit instead of the home-made protocol. The values of the NanoDrop are shown below. Both the yield and purity were better than before.
ng/µl 260/280 260/230 K823016 405.4 1.93 2.29 C0060 110.2 1.45 1.64 R0080 71.3 2.28 2.16 C0076 199.2 1.30 0.97 C0077 200.3 1.94 2.17 This week we also digested the bricks C0067, C0077, C0080, K823016, P0340 and C0060 with SpeI en EcoRI. We put the digestions on a gel and all bricks showed the correct bands (one for the insert and one for the vector). We excised the bands and purified them with a kit.
Order: ladder - K823016 - P0340 – C0080 – C0077 – C0060 – C0076
We cut the plasmid containing brick I719005 with SpeI and PstI, dephosphorylated the vector and did a pcr purification. We used the NanoDrop and measured a concentration of 34,2 ng/µl. -
Week 6: Our very first FFL-biobrick!
We started this week by doing the following digestions:
- B0015 with EcoRI and XbaI
- K608002 with SpeI and PstI
- B0034 with SpeI and PstI
Afterwards we treated the digestion mixtures with phosphatase and used a kit for pcr purification. The NanoDrop gave us these values afterwards:
ng/µl 260/280 260/230 B0015 A 73 1.7 0.82 B 35.6 1.96 1.09 C 56 1.83 1.09 D 43.6 1.91 1.05 E 70.9 1.74 0.84 K608002 37.6 2.04 1.5 B0034 38 2.01 2.02 Our new brick with GFP + terminator (K082003+B0015) was cut with XbaI and PstI and put on a gel. The insert was extracted and purified with a gel extraction kit.
Order: ladder - explanation
Afterwards we did following ligations:
- C0076 (insert) + B0015 (vector)
- C0077 (insert) + B0015 (vector)
- C0080 (insert) + B0015 (vector)
- K823016 (insert) + B0015 (vector)
- (K082003+B0015; insert) + K608002 (vector)
- (K082003+B0015; insert) + B0034 (vector) Ampicilline resistant
- P0340 (insert) + I719005 (vector) Ampicilline resistant
- C0060 (insert) + B0015 (vector)
- R0087 (vector; cut with EcoRI and PstI) + (P0340 + I719005; A; insert)
- R0087 (vector; cut with EcoRI and PstI) + (P0340 + I719005; B; insert)
- B0034 (vector)+ (C0076 +B0015; insert)
- R0087 (vector; cut with SpeI and PstI)+ (K082003+B0015 + B0034; insert)
- R0080 (vector) + (K082003+B0015 + B0034; insert)
- K398326 (vector) + P0340 (insert)
- R0087 (vector; cut with EcoRI and PstI) + EBF 4
We transformed inoue cells with the ligation products, according to the ‘transformation kit protocol’. Two of the plates contained ampicilline, the other ones chloramfenicol. We incubated them overnight at 37°C. The next day all of the plates contained colonies, except for the one containing C0060+B0015. Therefore we used the same ligation product again to transform some new inoue cells. This time the transformation worked. We inoculated one colony of each plate in 4 ml LB in the presence of the correct antibiotics. Unfortunately only in 3 of the tubes bacteria had grown. We inoculated a colony from the failed plates again. The tubes with growth were used for plasmid extraction, the result is displayed below:
ng/µl 260/280 260/230 K082003+B0015+K608002 203.2 1.94 2.48 C0077+B0015 136.4 1.97 2.55 P0340+I719005 206.9 1.94 2.46 We used 10 µl of each to do a digestion with EcoRI and PstI and put them on a gel to check if the correct inserts are present, which was not the case.
The next day all tubes contained grown cells, so this time the inoculation didn’t fail. We did a plasmid extraction and NanoDrop:ng/µl 260/280 260/230 Enzymes Correct insert? 1 K823016 + B0015 89.2 1.69 1.25 XbaI +PstI 2 (K082003+B0015) + B0034 38.8 3.04 0.62 XbaI +PstI Ok 3 C0076 +B0015 47 2.26 1.25 XbaI +PstI Ok 4 P0340 + I719005 A 37.6 3.28 0.74 EcoRI + PstI Ok 5 B 30.4 3.28 1.30 EcoRI + PstI Ok 6 C0077 + B0015 A 125.3 2.27 1.86 XbaI +PstI 7 B 24.6 5.92 1.2 XbaI +PstI 8 C0060 + B0015 31.3 3.42 0.99 XbaI +PstI 9 (K082003+B0015)+ K608002 A 44.9 2.32 1.82 XbaI +PstI 10 B 31.2 4.34 0.87 XbaI +PstI 11 C0080 + B0015 54 2.27 0.56 XbaI +PstI Afterwards we digested the vectors with the enzymes indicated in the table. We put them all on a gel but only four had the correct bands (see last column). These plasmids were sent for sequencing.
Order: ladder - 4 - 5 – 9- 10 – 6 – 7 - 8 - 11 - 3 - 1 - 2
We took some cells containing our new brick (GFP + terminator) out of the -80°C and put them in 4 ml LB with chloramphenicol. After plasmid extraction (using the kit), we became the values shown below. We sent this plasmids for sequencing.
ng/µl 260/280 260/230 K082003+B0015 A 101.3 1.98 2.67 B 105.3 1.96 2.42 We did the same thing for the 4 ligations that succeeded. After plasmid extraction we got the following concentrations:
ng/µl 260/280 260/230 (K082003+B0015) + B0034 25.8 2.05 2.12 P0340 + I719005 A 59.2 1.95 1.68 B 72.8 1.94 1.99 C0076 + B0015 117.7 1.87 1.35 We digested the vectors containing R0078, R0080 and B0034 with SpeI and PstI. The vector with R0078 was also cut once with EcoRI and PstI. We dephosphorylated the vectors and did a clean-up with the pcr purification kit.
We extracted the four correct bands out of the gel and purified them. Afterwards we did the following ligations:We digested the vector with K398326 with SpeI and PstI and EBF 4 with EcoRI and PstI. Both will be used in the napkin model. The first one was dephosphorylated and pcr purified with a kit. The following ligations were done:
We transformed inoue cells with those seven ligation products and we also transformed some cells again with the remaining ligation product of K823016 + B0015; C0077 + B0015; (K082003+B0015)+ K608002; C0080 + B0015 (these are the ones that didn’t show good bands on gel). So in total we did eleven transformations. All plates showed some growth and we inoculated four colonies from each plate in LB in the presence of the correct antibiotics.