Team:NTNU-Trondheim/Notebook

SulFind 2021

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Notebook

This page contains our day-to-day work in the laboratory. As we worked on two approaches for the main part of the summer, the documentation of the laboratory work is divided into the Heme protein approach and the Sulfide-responsive transcriptional repressor approach. In the fall of 2021 we performed work related to Improvement of an existing part.


Week 27

07/07/21

  • Prepared and autoclaved laboratory equipment
  • Prepared LB medium

08/07/21

Heme protein approach:

  • Prepared stock solution with Myoglobin and made aliquots with concentration 0.1 g/L.

Week 28

12/07/21

Sulfide-responsive transcriptional repressor approach:

  • Diluted primers for cloning to a final concentration of 10 μM.

13/07/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the efficiency of the supercompetent DH5-alpha E. coli cells, and incubated them overnight.
  • PCR-amplified the pENZ004 plasmid for Gibson assembly.
  • Analyzed the samples from PCR on an agarose gel.
  • Removed the WT plasmid from the PCR samples with Dpn1 enzyme digest.

Heme protein approach:

  • Prepared stock solution with Cy3 NHS ester.
  • Prepared stock solution of Potassium Phosphate buffer.
  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

14/07/21

Sulfide-responsive transcriptional repressor approach:

  • Suspended DNA sequences from IDT.
  • Performed PCR purification, Gibson assembly and heat shock transformation.
  • Counted the number of colonies from the competent cell test from yesterday.

Heme protein approach:

  • Purified labeled myoglobin from 13/07/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution and (H2S) dilutions.

15/07/21

Sulfide-responsive transcriptional repressor approach:

  • Transferred cells to LB amp medium and incubated overnight.

16/07/21

Sulfide-responsive transcriptional repressor approach:

  • Isolated plasmid DNA from the transformed DNA. NB! At step 10 only 30 μL EB buffer should be added, instead of 30 μL as the protocol states.
  • Prepared Glycerol stock.

Week 29

19/07/21

Sulfide-responsive transcriptional repressor approach:

  • Performed Gibson assembly and transformation for design 2 fragments.
  • Performed restriction digest and gel analysis.
  • Tested the whole cell sensor with (H2S) and design 1 (A, B & C) - part 1. Frozen transformed bacteria were thawed on ice and added to a 5 ml solution of LB(Amp). Incubated overnight.

20/07/21

Sulfide-responsive transcriptional repressor approach:

  • Performed Gibson assembly and transformation for design 2 fragments.
  • Sent to Eurofins for Sanger sequencing (Miniprepped DNA was dried out overnight, to make it concentrated enough for sequencing. Template and sequencing primers were mixed in accordance with Eurofins protocol and sent for sequencing).
  • Transformation with design 2A (gibson assembly product) - part 1.
  • Tested the whole cell sensor with (H2S) and design 1 (A, B & C) - part 2. Bacterial solutions from 19/07/21 were transferred to LB solution and incubated overnight.

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

21/07/21

Sulfide-responsive transcriptional repressor approach:

  • Performed transformation with design 2A
  • Sent 2BS-C1, 2BS-C2, 2BL-C1, 2BL-C2 and 2AL to Eurofins for Sanger sequencing.
  • Tested the whole cell sensor with (H2S) [2000,85 M] and design 1 (A, B & C) - part 3. Diluted bacterial solution and added analytes. Incubated it for 17 hours

Heme protein approach:

  • Purified labeled myoglobin from 20/07/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [5 mg/L] and (H2S) dilutions [0.05-0.3 umol/L]. Measured fluorescence with Tecan.

22/07/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) [2000,85 M] and design 1 (A, B & C), measured fluorescence - part 3;
  • Froze design 2 cells, mini prepped and sent to Eurofins for sanger sequencing.

23/07/21

Heme protein approach:

  • Performed emission/excitation scan measurements on the Tecan to identify emission/excitation peaks of the Cy3 fluorophore used in the heme approach. Used the labeled myoglobin from 21/07/21.

25/07/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH). Frozen transformed bacteria were thawed on ice and added to a 5 ml solution of LB(Amp). Incubated overnight.

Week 30

26/07/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH). Bacterial solutions from the previous day were transferred to LB solution and incubated overnight.

    Heme protein approach:

    • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

27/07/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH) and design 1 (A, B & C). Diluted bacterial solution and added analytes. Incubated it for 17 hours.

Heme protein approach:

  • Purified labeled myoglobin from 26/07/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [5 mg/L] and (H2S) dilutions [0, 1, 5, 10, and 25 umol/L]. Measured fluorescence with Tecan.

28/07/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH) and design 1 (A, B & C), measured fluorescence.

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

29/07/21

Sulfide-responsive transcriptional repressor approach:

  • Froze bacteria, mini prepped and sent design 2A to Eurofins for Sanger sequencing.

Heme protein approach:

  • Purified labeled myoglobin from 28/07/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [25 mg/L] and (H2S) dilutions [0.25-1.5 umol/L]. Measured fluorescence with Tecan.

01/08/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH) and design 1A,1B,1C, 2AS, 2AL, 2BS, 2BL. Frozen transformed bacteria were thawed on ice and added to a 5 ml solution of LB(Amp). Incubated overnight.

Week 31

02/08/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH) and design 1A,1B,1C, 2AS, 2AL, 2BS, 2BL. Bacterial solutions from the previous day were transferred to LB solution and incubated overnight.
  • Colony PCR of 2AL and 2AS

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

03/08/21

Sulfide-responsive transcriptional repressor approach:

  • Colony PCR - the results indicated that 1A, 1B, 1C and 2A do not work.
  • Tested the whole cell sensor with (H2S) (GSSH) and design 2BS. Diluted bacterial solution and added analytes. Incubated it for 17 hours.

Heme protein approach:

  • Purified labeled myoglobin from 02/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [5 mg/L] and (H2S) dilutions [0-1.5 umol/L]. Measured fluorescence with Tecan.

04/08/21

Sulfide-responsive transcriptional repressor approach:

  • Tested whole cell sensor with (H2S) (GSSH) and design 2BS, measured fluorescence.

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

05/08/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) (GSSH) and design 2BS, measured fluorescence again.

    Heme protein approach:

    • Purified labeled myoglobin from 04/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [5 mg/L] and (H2S) dilutions [0-1.5 umol/L]. Measured fluorescence with Tecan.

06/08/21

Sulfide-responsive transcriptional repressor approach:

  • Tested the whole cell sensor with (H2S) and design 2BS, measured fluorescence.

08/08/21

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

Week 32

09/08/21

Heme protein approach:

  • Purified labeled myoglobin from 08/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [5 mg/L] and (H2S) dilutions [0, 0.001, 0.01, 0.1, 1, 10, uM]. Measured fluorescence with Tecan in addition to a kinetic study.
  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

10/08/21

Heme protein approach:

  • Purified labeled myoglobin from 09/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [5 and 25 mg/L] and (H2S) dilutions [0, 0.001, 0.01, 0.1, 1, 10, uM]. Measured fluorescence with Tecan.
  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

11/08/21

Heme protein approach:

  • Purified labeled myoglobin from 10/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [1, 5, 10, 20, 40, mg/L] and (H2S) dilutions [0.1 uM]. Measured fluorescence with Tecan in addition to a kinetic study.

15/08/21

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

Week 33

16/08/21

Heme protein approach:

  • Purified labeled myoglobin from 15/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [1, 5, 10, 20, 40 mg/L] and (H2S) dilutions [1 uM]. Measured fluorescence with Tecan in addition to a kinetic study.
  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

17/08/21

Heme protein approach:

  • Purified labeled myoglobin from 16/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [1, 5, 10, 20, 40 mg/L] and (H2S) dilutions [0.1 uM]. Measured fluorescence with Tecan.
  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

18/08/21

Heme protein approach:

  • Purified labeled myoglobin from 17/08/21, measured myoglobin concentration and labeling ratio, prepared labeled protein dilutions and (H2S) dilutions. Tested two different kinetic studies. The first with labeled protein [1, 5, 10 and 20 mg/L] and (H2S) [0.1 uM], and another with labeled protein [5 mg/L] and (H2S) [0.001, 0.01, 1.0 and 2.0 uM].

Week 36

10/09/21

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

11/09/21

Heme protein approach:

  • Purified labeled myoglobin from 10/09/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [10 mg/L] and (H2S) dilutions [1 uM].

    Performed a kinetic study where different time intervals ( 3 sec, 30 sec, 1 minute and 5 minutes).

    Improvement of an existing part:

    • Started the cloning process (PCR, PCR gel analysis and Dpn1 enzyme digest).

Week 37

13/07/21

Improvement of an existing part:

  • Picked two colonies of each sample from 11/07/21, and inoculated in LB/amp overnight.

14/07/21

Improvement of an existing part:

  • Transferred bacteria cell culture into LB/amp, incubated from 08:45 to 12:45. Diluted IPTG to 40 and 400 uM in bacteria culture, and incubated 3-5 hours in 37 degrees. Tested bacteria with IPTG and a control (bacteria without IPTG) with Tecan.
  • Miniprep and sequencing.

15/07/21

Improvement of an existing part:

  • Made negative control culture for UV and fluorescence measurements.

16/07/21

Improvement of an existing part:

  • Measured fluorescence intensity and recorded UV images of grown cultures.

Week 40

05/10/21

Heme protein approach:

  • Labeled myoglobin with Cy3 NHS ester, incubated overnight.

06/10/21

Heme protein approach, implemented in Lab on a Chip:

  • Purified labeled myoglobin from 05/10/21, measured myoglobin concentration and labeling ratio, prepared labeled protein solution [10 mg/L] and (H2S) dilutions [0.1, 0.5 and 1 uM]. Lab on a Chip was used to measure the fluorescence.


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