Team:Bielefeld-Germany/Labjournal/week21

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(Difference between revisions)
(Sunday September 23rd)
(Sunday September 23rd)
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** For collaboration with [http://2012.igem.org/Team:University_College_London UCL London]: Cultivation of ''E. coli'' KRX with <partinfo>BBa_K729006</partinfo> and ''E. coli'' KRX without plasmid as negative control
** For collaboration with [http://2012.igem.org/Team:University_College_London UCL London]: Cultivation of ''E. coli'' KRX with <partinfo>BBa_K729006</partinfo> and ''E. coli'' KRX without plasmid as negative control
*** Settings: 250&nbsp;mL flasks without baffles, final volume: 60&nbsp;mL, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#LB_medium LB-medium], 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, durance: 12&nbsp;hours.
*** Settings: 250&nbsp;mL flasks without baffles, final volume: 60&nbsp;mL, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#LB_medium LB-medium], 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, durance: 12&nbsp;hours.
 +
*** Settings: 1&nbsp;L flask without baffles, final volume: 200&nbsp;mL, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#LB_medium LB-medium], 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, durance: 12&nbsp;hours.
*** We determined the OD<sub>600</sub> for a growth kinetics (see figure X).
*** We determined the OD<sub>600</sub> for a growth kinetics (see figure X).
** Cultivation of ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo> for comparison.
** Cultivation of ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo> for comparison.

Revision as of 18:23, 25 September 2012


Contents

Week 21 (09/17 - 09/23/12)

Monday September 17th

  • Team Cultivation & Purification:
    • Cell disruption of fermentation of E. coli Rosetta-Gami 2 with BBa_K863012 via high-pressure homogenization and purification via Ni-NTA column.
Activity of ECOL measured via oxidized ABTS at OD420 in relation to a MeOH gradient.
Activity of ECOL measured via oxidized ABTS at OD530 in relation to a MeOH gradient.
  • Team Activity Tests:
    • Another day full of measurements is over! Today we measured some more samples from Team Immobilization: check their labjournal. As well we went on with characterizing our laccases. ECOL was analyzed regarding its behavior in different concentrations of MeOH and acetonitrile. The activity was measured with concentrations of 2-16 µL, respectively. Since ABTS is changing it´s emission spectrum when being in contact with methanol we also detected the OD530. As expected ECOL is happier when only little MeOH is in its environment. We did the same measurements with acetonitrile and got the same result: the fewer acetonitrile, the better for ECOL. Again, we will hand over these results to Team Substrate Analytics.
Activity of ECOL measured via oxidized ABTS at OD420 in relation to a acetonitrile gradient.
Activity of ECOL measured via oxidized ABTS at OD530in relation to a acetonitrile gradient.
    • Also another measurement session started today. Team Cultivation lend their purified laccases from TTHL and BHAL produced with constitutive promoters to us. SO we rebuffered them and checked the activity with our standard protocol. And the results were good! In both cases we could detect activity in each fraction that Team Cultivation gave us (see Fig. 1 and Fig. 2). We are happy to have more laccases, but we are not sure, if we can pay enough attention to characterize them. We are trying our best, but BPUL and ECOL come first.
Activity of TTHL measured via oxidized ABTS at OD420, showing activity in each fraction after prufication.
Activity of BHAL measured via oxidized ABTS at OD530, showing activity in each fraction after prufication.

Tuesday September 18th

  • Team Cellulose Binding Domain:
    • Primer arrived:
    • Gradient-PCR with the CBDcex(T7)+GFP_His-plasmid and S3N10-CBDcex_RV and S3N10-GFP_FW primer-mix. the right product appeared only at 66,5°C; Lower: wrong product; Higher: no product
      • Following PCR at 66,5°C(50µL) had the wrong product (main product at 2 kbp); could be because of a different template concentration or not exact temperature.
      • Alternativly took the 20 µL-tube with right product and added 0,5 µL DpnI for over-night digestion.
    • Gradient-PCR with CBDcex(T7)+GFP_His-template and a CBDcex_Freiburg- + GFP_Freiburg_compl-primer-mix to amplify the whole fusion-protein and get rid of the His-tag.
    • Gradient-PCR with CBDclos(T7)+GFP_His-template and a CBDclos_Freiburg- + GFP_Freiburg_compl-primer-mix to amplify the whole fusion-protein and get rid of the His-tag.
  • Team Activity Tests:
Fig. 1: Measurements of ECOL in a CuCl gradient of 0,1 to 0,7 mM. The activity was measured via oxidized ABTS at OD420, (n=4).
    • Today we measured the activity of our ECOL laccase in a CuCl gradient to check whether another concentration than the one we usually incubated the laccases with might be more optimal. We chose a gradient from 0,1 mM to 0,7 mM (with an increment of 0,1) and measured the incubated samples as usual in 100 mM sodium acetate buffer, 0,1 mM ABTS (ad 200 µL H2O), respectively. Fig. 1 shows that the chosen concentration do influence ECOL, 0,4 mM seems like the best choice for incubation before the activity measurments. Thus the spectrum of OD420 maxima is not immensely wide so that ECOL seems to be satisfied with any CuCl concentration.
Fig. 2: Activity measurements of TTHL via OD420.
    • Additionally we measured the activity of TTHL without constitutive promoter at 25°C and in sodium acetate buffer (pH 5). TTHL finally also shows some activity, our favorite fractions of Team Cultivations output are number 1 and 2. Unfortunately TTHL couldn´t be indentified correctly via MALDI yet so that we will have to wait with further characterizing until we get the go through the MALDI results.
    • And another big suprise was the activity test of BPUL. After Team cultivation fermented it in a 6 L fermentation and purified our lovely enzyme, we did the standardized activity measurement protocol overnight. And have a look at our great results in fig. 6! We clearly have a good activity of BPUL reaching its saturation after half an hour. A fast reaction like this is comparable to the store bought laccase TVEL0. We are happy and will rebuffer as much as we can from fraction 4 which seems to have most of the Protein. Also we are going to determine the protein concentration. This is going to be awesome!
      Fig. 2: Activity measurements of BPUL via OD420. Different fractions from Team Cultivations purification were analyzed and they all showed activity.
  • Team Substrate Anayltics:
    • We analyze Ethin estradiol and estradiol degradation with the Spectrofluorophotometer. The measure showed that the degradation product are detectable but the results weren't clear. We decide to do this measurement again to have better results so a new degradation of the two substrates were set.








Wednesday September 19th

  • Team Cellulose Binding Domain:
    • Stopped over-night-DpnI-digestion of S3N10-clos-GFP_his PCR-product and cleaned up in gel. Ligated the PCR-product with itself via Blunt-end-ligation and transformed it into KRX. Plated on Select-Agar.
    • Gradient-PCRs of the CBDcex(T7)+GFP_His-plasmid and a CBDclos(T7)+GFP_His-plasmid (unsequenced) with the CBDcex_Freiburg- and GFP_Freiburg-compl- and the CBDclos_Freiburg- and GFP_Freiburg_compl primers, respectively. The CBDcex-setup showed no correct product, but the CBDclos-setup had the right bands at temperatures from 57°C to 64°C; Merged the correct fractions and cleaned them up through the gel.
    • Digested the cleaned-up product with SpeI and XbaI and DpnI.
    • Ligated BBa_J61101 and CBDclos_Freiburg with GFP_Freiburg and transformed it into KRX.
  • Team Activity Test:
    • What a day! The deadline is coming closer and we still had (!) a lot of measurements to do, so today was chosen to be a day full of important measurements. First we measured the activity of the TVEL0 and BPUL in a CuCl gradient. As well we examined what our BPUL laccase does when its supposed to work in different pHs. BPUL was additionally characterized regarding its behavior in different concentrations of MeOH and acetonitrile. This output is of course most interesting for Team Substrate Analytics.
      Comparison of activity of ECOL in sodium acetate buffer and Britton-Robinson buffer, measured at OD420 respectively.
      Since we already compared the behaviour of TVEL0 in the environment of sodium acetate buffer (pH 5) and Britton-Robinson buffer (pH 5), respectively, we also compared BPUL and ECOL in those buffers. ECOL is not impressed by the two different buffer systems and thus not influenced in it´s activity.
BPUL laccase activity measured via ABTS and OD420 under usage of different pHs of sodium actate buffer. The results show a high activity at pH 5.
Negative control for pH measurements using ABTS. ABTS is oxidized through havy metals like Cu, which we are using for laccase activity tests. At different tested pHs ABTS gets oxidized but not very fast allowing us to leave it outer consideration.
BPUL laccase activity measured with different concentration of CuCl showing that an incubation with 0.6 mM CuCl is optimal for the laccae activity, but higher CuCl concentrations seem to interfere in the activity.
Negative control for analyzing the oxidization of ABTS by different CuCl concentrations. An effect on the oxidization of ABTS is clearly distinguishable, but between the applied concentrations there is no difference in OD420.
Characterization of the activity of BPUL with sodium acetate buffer and Briton-Robinson Buffer at pH 5. Using the Briton-Robinson Buffer the saturation of BPUL activity is reached slower but stays constant. With sodium acetate buffer the maximum is reached considerably earlier.
BPUL activity under different concentrations of acetonitrile. Acetonitrile has a small impact on the activity but indicates a decrease in activity the more actetonitrile is used.
BPUL activity under different concentrations of MeOH. MeOH seems to have no impact on the activity using this small amounts of it.



Thursday September 20th

  • Team Fungal and Plant Laccases :
    • Our primers for GFP, tvel35, tvel5 and BBa_K500002 arrived. We did PCR on GFP with the new primers for cloning in our shuttle vector.
  • Team Cellulose Binding Domain:
    • No Colonies on the S3N10-CBDclos(T7)+GFP_His-transformations-dish with the blunt-end ligation; reason: forgotten to phosphorylate the PCR-product.
    • Two colonies on the CBDclos+GFP-dish with the constitutive J23100 + J61101 promoter. But none of them is glowing. And them shouldn't ... used the wrong selection-agar (since J61101 carries pSB1A2-backbone with AMP-resistence.
  • Team Activity Tests:
Temperatur measurements of ECOL at 10°C and 25°C. Measurements were done via optical density 420 nm.
Temperatur measurements of BPUL at 10°C and 25°C. Measurements were done via optical density 420 nm.
    • Today we started another exciting measurement. Today was all about temperatures. Through our cooperation with the clarification plant we found out that the waster water here in Germany has a minimal temperature of 10°C throughout the year. We used this value together with our previous measurements at 25°C to complete the temperature gradient of our measurements. Eventhough ECOL shows an increased activity at 10°C, it does reach a comparable OD value as the samples measured at 25°C. The increased activity is expressed by achieving the maximal OD420 slower than the laccase measured at 25°C. This seems like good news for Team Modeling. On the other hand BPUL shows nearly the same great activity at 10°C as at 25°C. Of course the reaction is a bit slower, but nevertheless the saturation is reached after 3 hours.
  • Team Substrate Analytics:
    • Degradation measure from Ethinyl estradiol and Estradiol with the Spectrofluorophotometer. Degradation was from the 18th September

Friday September 21st

  • Team Fungal and Plant Laccases:
    • Digest of BBa_K863204 and GFP PCR product with AarI and ligation. Transformation in E. coli KRX cells.
  • Team Cellulose Binding Domain:
    • Transformated BBa_J61101 with CBDclos+GFP again and plated on the right selection-agar this time!
ECOL activity with different concentration of ABTS.
BPUL activity with different concentration of ABTS.
  • Team Activity Tests:
    • Another part of the characterization of our ECOL and BPUL laccase was to measure their activity when exposed to different concentrations of ABTS. We choose an exponential gradient of 2 µL to 16 µL and measured with the usual laccase concentration of 0,003 mg/mL and 100 mM sodium acetate buffer (ad 200 µL H2O). Unfortunately the measurements that contained 16 µL ABTS could not be measured completely because the OD420 exceeded the maximal value that Tecan was able to measure. The results of ECOL (see. Fig. 1) show as well an exponential increase in the OD420 correlated to the ABTS concentrations, respectively. The negative control was chosen to contain the maximum of 16 µL ABTS in buffer and water that was incubated with CuCl2.

Saturday September 22nd

  • Team Fungal and Plant Laccases:
    • We did colony PCR on the transformed cells from day before. The results show that almost all colonies have the insert GFP. So we picked three of the positive colonies and plated each on five LB + CM plates, because ee need much vector for the transformation in Pichia pastoris.
  • Team Cellulose Binding Domain:
    • Once again two colonies on the agar-dish: can not say if they have a green glow...
      • picked the colonies and them put into AMP-LB-Medium.
    • Also made a flask with 10 mL LB-Medium and cells with the CBDcex(J61101)+GFP_His-plasmid

Sunday September 23rd

  • Team Cellulose Binding Domain:
    • All cultures did not show any sign of GFP-glow under uv-light.
    • Gradient-PCR of BBa_K863104 with S3N10_Cex_compl- and S3N10_GFP-primermix to generate a 13 amino acid-linker between the cellulose binding domain and the GFP.
    • Transformated CBDcex(T7)+GFP_His and CBDclos(T7)+GFP_His into BL21, to test if the problem is in the expression-system.
  • Team Cultivation & Purification:
Figure x: Growth kinetics of flask cultivation of E. coli KRX with BBa_K729006 and without plasmid as negative control using LB-medium and of E. coli KRX with BBa_K863005 using autoinduction medium. Further parameters: final volume of 60 mL, 60 µg/mL chloramphenicol, 37 °C, durance: 12 hours.
    • For collaboration with UCL London: Cultivation of E. coli KRX with BBa_K729006 and E. coli KRX without plasmid as negative control
      • Settings: 250 mL flasks without baffles, final volume: 60 mL, LB-medium, 60 µg/mL chloramphenicol, 37 °C, durance: 12 hours.
      • Settings: 1 L flask without baffles, final volume: 200 mL, LB-medium, 60 µg/mL chloramphenicol, 37 °C, durance: 12 hours.
      • We determined the OD600 for a growth kinetics (see figure X).
    • Cultivation of E. coli KRX with BBa_K863005 for comparison.
      • Settings: 250 mL flasks without baffles, final volume: 60 mL, autoinduction medium, 60 µg/mL chloramphenicol, 37 °C, durance: 12 hours.
    • Made SDS-Page of the cultivation from 09/15.
Figure 1: First fermentation of E. coli KRX with BBa_K863012 (09/15). 6 L in fermenter NFL22, 37 °C, pO2 of 30 % for 12 hours., LB-medium with 60 µg/mL chloramphenicol and 300 µg/mL ampicillin. Purification of the supernatant via His-trap column. The red arrow shows TTHL in lane 9-11 (elution 1-3).