Difference between revisions of "20.109(S23):M2D4"

From Course Wiki
Jump to: navigation, search
(Created page with "<div style="padding: 10px; width: 820px; border: 5px solid #434a43;"> {{Template:20.109(S23)}} ==Introduction== Image:Fa16 M2D7 tet repression schematic.png|thumb|right|5...")
 
Line 102: Line 102:
 
*Why is it important to wash the secondary antibody from the coverslip before imaging?
 
*Why is it important to wash the secondary antibody from the coverslip before imaging?
 
*What stain is used following secondary antibody?  What cellular component is stained in this step?  And why is this useful?
 
*What stain is used following secondary antibody?  What cellular component is stained in this step?  And why is this useful?
 +
 +
==Navigation links==
 +
Next day: [[20.109(S23):M2D5 |Analyze ICP-OES data]] <br>
 +
Previous day: [[20.109(S23):M2D3 |Sequence clones and transform into yeast cells]] <br>

Revision as of 22:23, 31 January 2023

20.109(S23): Laboratory Fundamentals of Biological Engineering

Sp23 banner image v2.png

Spring 2023 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Drug discovery        M2: Protein engineering        M3: Project design       

Introduction

Schematic of aTc induction of tet promoter.
In contrast, expression of the gene encoding dCas9 within pdCas9 is regulated by an inducible promoter (pLtetO-1). An inducible promoter is 'off' unless the appropriate molecule is present to relieve repression. In the case of the CRISPRi system, expression of the gene encoding dCas9 is inhibited due to the use of a tet-based promoter construct. Tet is shorthand for tetracycline, which is an antibiotic that inhibits protein synthesis through preventing the association between charged aminoacyl-tRNA molecules and the A site of ribosomes. Bacterial cells that carry the tet resistance cassette are able to survive exposure to tetracycline by expressing genes that encode an efflux pump that 'flushes' the antibiotic from the bacterial cell. To conserve energy, the tet system is only expressed in the presence of tetracyline. In the absence of tetracycline, a transcription repressor protein (TetR) is bound to the promoter upstream of the tet resistance cassette genes. When tetracycline is present, the molecule binds to TetR causing a confirmational change that results in TetR 'falling off' of the promoter. In the CRISPRi system, the tet-based promoter construct upstream of the gene that encodes dCas9 is 'off' unless anhydrotetracyline (aTc), an analog of tetracyline, is added to the culture media. Why is it important to use an analog rather than the actual antibiotic?

Taken together, the sgRNA_target is constitutively transcribed and, thereby, always present. The dCas9 protein is only present when aTc is added. Thus, gene expression is only altered when aTc is present. In this, when dCas9 is expressed it forms a complex with the sgRNA_target. The sgRNA target then 'seeks out' the target within the host genome. When the targeted sequence is recognized, the complex binds and acts as a 'roadblock' by prohibiting RNAP access to the sequence. Because the targeted gene is not able to be transcribed, the protein encoded by that gene is not synthesized. In our experiments, we hypothesize that the absence of specific proteins, or enzymes, involved in anaerobic fermentative metabolism will increase the yield of ethanol.

Protocols

Part 1: Examine psgRNA_target sequencing results

Your goal today is to analyze the sequencing data for you two potential mutant psgRNA clones - two independent colonies from your cloning reaction - and then decide which colony to proceed with for the CRISPRi engineering of the E. coli MG1655 fermentation pathway.

Retrieve psgRNA_target sequence results from Genewiz

  1. Your sequencing data is available from Genewiz. For easier access, the information was uploaded to the [20.109(S22):Class_data Class Data tab].
  2. Download the zip folder with your team sequencing results and confirm that there are 8 files saved in the folder.
  3. For each sequencing reaction, you should have one .abi file and one .seq file.
  4. Open one of the .abi files.
    • This file contains the chromatogram for your sequencing reaction. Scroll through the sequence and ensure that the peaks are clearly defined and evenly spaced. Low signal (or peaks) or stacked peaks can provide incorrect base assignments in the sequence.
  5. Open one of the .seq files.
    • This file contains the base assignments for your sequencing reaction. The bases are assigned by the software from the chromatogram sequence.
    • The start of the a sequencing reaction result often contains several Ns, which indicates that the software was unable to assign a basepair.

In your laboratory notebook, complete the following:

  • Given the chromatogram result, why might the software assign Ns in the start of the sequence?
  • Visually inspect the chromatograms for all of your sequencing results.
    • Do the peaks appear clearly defined or is there overlap? What might this indicate about the quality of your sequencing results?
    • Do the peaks extend above the background signal? What might this indicate about the quality of your sequencing results?

Confirm sgRNA_target sequence in psgRNA_target using SnapGene

You should align your sequencing data with a known sequence, in this case the gRNA target sequence you selected, to identify any unintended base changes that may have occurred. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using SnapGene are below. Please feel free to use any program with which you are familiar.

  1. Generate a new DNA file that contains the sgRNA oligo you designed on M2D2. This file should contain only the target sequence you selected and the dCas9 tag sequence.
  2. Generate an additional new DNA file that contains the results from the sequencing reaction completed by Genewiz.
    • For each sequencing result you should generate a distinct new DNA file. Remember you should have a forward and reverse sequencing result for each of your clones!
    • Paste the sequence text from your sequencing run into the new DNA file window. If there were ambiguous areas of your sequencing results, these will be listed as "N" rather than "A" "T" "G" or "C" and it's fine to include Ns in the query.
    • The start and end of your sequencing may have several Ns. In this case it is best to omit these Ns by pasting only the 'good' sequence that is flanked by the ambiguous sequence.
  3. To confirm the sgRNA sequence in your clones, open one of the forward sequencing results files generated in the previous step.
    • Select 'Tools' --> 'Align to Reference DNA Sequence...' --> 'Align Full Sequences...' from the toolbar.
    • In the window, select the file that contains the sgRNA oligo sequence and click 'Open'.
  4. A new window will open with the alignment of the two sequences. The top line of sequence shows the results of the sequencing reaction and the bottom line shows the oligo you designed.
    • Are there any discrepancies or differences between the two sequences? Scroll through the entire alignment to check the full sequencing result and note any basepair changes.
  5. Follow the above steps to examine all of your sequencing results. Remember: you used a forward and a reverse primer to interrogate both potential psgRNA_target plasmids.
  6. From the alignments, determine which psgRNA_target has the correct sgRNA_target sequence.
    • If both clones contain the correct sequence choose either co-transformant to use to test ethanol / acetate yield. If only one is correct, then this is the co-transformant you will use. If neither of your plasmids carry the appropriate insert, talk to your Instructor.

In your laboratory notebook, complete the following:

  • Attach a screenshot for each alignment.
  • Record which clone contains the correct sgRNA_target sequence.

Part 2: Perform antibody staining for γH2AX assay

Complete primary staining steps

Immunofluorescence staining chamber
  1. Obtain your 12-well plates from the front laboratory bench.
  2. Gather an aliquot of 1 X TBS from the front laboratory bench.
    • Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) in 1X TBS in a micro centrifuge tube using the 10% Triton stock is at the front laboratory bench.
    • Prepare 2.5 mL solution of 1% BSA (v/v) in 1X TBS in 15 mL conical tube. 10% BSA stock is at the front bench.
      • One of the preparations will be the blocking solution used in Step #8 and the other preparation will be used in Step #9 for the primary antibody solution.
  3. Obtain a staining chamber from the front bench and add a damp paper towel to each side of the parafilm.
    • Label the parafilm with experimental details.
  4. Obtain a fine gauge (26 3/8) needle and a pair of tweezers from the front laboratory bench.
    • Carefully press the tip of the needle against the benchtop to bend it into a right angle such that the beveled side of the needle is the interior angle.
  5. Use the 'hook' created with the needle to lift the coverslip from the bottom of the well, then use the tweezers to 'catch' the coverslip.
    • Practice plates with coverslips will be available at the front laboratory bench.
  6. When you are confident with your ability to retrieve the coverslips from the wells, move one coverslip from each condition from your 12-well plates to the staining chamber. Cell-side UP!
    • The cell-side of the coverslip is the side that was facing up in the well of the 12-well plate.
  7. Quickly permeabilize the cells by adding 150 μL of the 0.2% Triton X-100/TBS solution to each coverslip and incubate for 10 min at room temperature.
  8. Aspirate the 0.2% Triton X-100/TBS solution and add 150 μL of BSA blocking solution to each coverslip, then incubate for 60 min at room temperature.
  9. With 15 min remaining of the blocking solution incubation, prepare the primary antibody.
    • Dilute the mouse anti-γH2AX antibody 1:1000 in the 1.2 mL aliquot of BSA blocking solution.
  10. Aspirate the block solution and add 150 μL of the diluted primary antibody solution to each coverslip before moving the next. Do not let the coverslips dry!
  11. Cover your staining chamber with the lid to incubate at room temperature.
  12. Incubate samples in the primary antibody solution for ~1 h.

Complete secondary staining steps

  1. Aspirate the primary antibody and wash each coverslip by pipetting 200uL of TBS to the top of the coverslip, then use pipet or aspirator to remove liquid.
    • Complete a total of 3 washes. At the final wash leave the liquid on the coverslip.
  2. Retrieve aliquot of diluted secondary antibody, Alexa Fluor 488 goat anti-mouse (diluted 1:200 in blocking solution) from front bench.
  3. Aspirate the wash liquid from one coverslip and immediately add 150 μL of the diluted secondary antibody to the coverslip.
    • Complete this step for each coverslip individually as it is important that the coverslips do not dry!
  4. Cover your coverslips to protect them from light.
  5. Incubate samples at 4 °C in the secondary antibody solution for ~1 h.
  6. Make sure to have TBS solution available before you start. Aspirate the secondary antibody solution off the coverslip and immediately add 150 μL of TBS. Do not let the coverslips dry out during this process.
  7. To complete the post secondary wash, add 150 μL of TBS per coverslip, let incubate at room temperature for 3 min covered, then aspirate.
  8. To add DAPI, dilute the DAPI stain 1:1000 in TBS and add 150 μL DAPI per coverslip.
  9. Incubate at room temperature for 10 min covered, then aspirate.
  10. Add TBS as in Step #2 for the final wash and leave for 3 min. Do not aspirate.
  11. Obtain glass slides from the front laboratory bench and label your slides with all of your experimental information and group name, add one drop (15 uL) of mounting media to the slide.
  12. Aspirate the final TBS wash and using tweezers place the coverslip cell-side down on the mounting media "spot" on the microscope slide. Try your best to avoid bubbles by slowly placing the coverslip over the mounting media.
    • The cell-side of the coverslip is the side that was facing up in the staining chamber.
  13. Complete Steps #5-6 for coverslips from all of the coverslips you stained.
  14. Add one small drop of nail polish to each side of your coverslip to seal it to the glass slide.

In your laboratory notebook, complete the following:

  • Why is it important to wash the secondary antibody from the coverslip before imaging?
  • What stain is used following secondary antibody? What cellular component is stained in this step? And why is this useful?

Navigation links

Next day: Analyze ICP-OES data

Previous day: Sequence clones and transform into yeast cells