Creating ORF fusions between GFP & LepA Gene

DAY 10, Oct. 10: CONFIRMATION OF GFP POSITIVE STRAINS AND INOCULATION OF

POSITIVE CANDIDATES INTO LIQUID MEDIA Purpose: To confirm we have genetically pure GFP positive isolates and start liquid

cultures for future analysis of plasmid DNA sequence and GFP protein expression

Procedure:

1. Lab partner pairs should have two plates from DAY 8. Each plate contains a re-streak of an original colony that was GFP-positive. Inspect plates to confirm GFP-positive colonies.

2. Record whether there is growth on each plate and record whether or not there are GFP- positive colonies.

3. Each student pick one GFP positive colony (If there is growth on both plates from Day 8, be sure that one colony is taken from each plate. If there is growth on only one plate or no growth on either plate, then consult with instructor to formulate a plan). Each GFP-positive colony should be transferred into one 15 mL sterile conical tube containing 1.5 mL of LB Amp. Incubate this tube at 30°C during the lab.

4. While waiting for these incubations prepare and label all of the tubes that you will need as described below. Also use the Gel Doc to take pictures of your streak plates.

5. From 1.5 ml tube take 0.2 ml and load it to the 1.8ml LB+Amp. Label this tube “NO IPTG”

6. From my own culture take 1 ml and added to the 9 ml LB Amp+ 6 ul IPTG. Label it “+ IPTG”

7. Incubate the conical tubes at 30°C for 24 hrs.

DAY 11, Oct.12: ISOLATION OF PLASMID DNA, PREPARATION OF PROTEIN GEL LYSATES; DNA SEQUENCING REACTION

Purpose: To isolate pPEM109 plasmid and total protein from liquid cultures containing the

GFP-positive transformant

Procedure:

1. PLASMID PURIFICATION: Resuspend the cells from your 1.8 mL culture from Day 10 labeled “No IPTG” (they will have settled out) and transfer 1.5 mL into a standard 1.5 mL eppendorf tube. Label this tube “Day 11 plasmid DNA” with your candidate number. Transfer another 1 mL from the conical tube into eppendorf tube 1.5 mL tube. Label this tube “- IPTG protein. Use your “Day 11 plasmid DNA” tube to isolate plasmid DNA according the procedure described in this manual on pages 14-15. And REMOVE 200 ul from the 1.8 ml culture to a new tube label it as “-IPTG Protein”.

2. Using your 9 mL culture from Day 10 (in the 50 mL conical tube, labeled “+IPTG”) transfer 0.2 mL into a Safe-Lock 1.5 mL tube (remember to resuspend settled cells prior to transfer). Label this tube “+IPTG protein”.

3. There should be ~9 mLs of bacteria culture remaining in one 50 mL conical tube (+IPTG) that should be collected in a tube that fits into the Bead Beating apparatus. These special tubes will be distributed to you. Label the bead beater tubes with your candidate number BOTH “+IPTG native.” Note that the tube capacity is less than 4 mL. To collect 4 mLs of cells from the remaining cultures you will fill up the tube, centrifuge the cells and refill the tube until you have collected all of the cells from the 4 mL culture into one cell pellet. These cell pellets will be stored at -20oC for future use.

4. You have a Safe-Lock tube from step 1 labeled “-IPTG protein” and a tube from step 2 labeled “+IPTG protein.” Collect the cells in these tubes by centrifugation, remove the supernatant and add 20 ul of sterile water (more or less depending on size of pellet). Resuspend the cells and add equal volume of 2X SDS sample buffer (with reducing agent). Mix by inversion. Ensure that the Safe-Lock tube is locked. Place samples in 100°C heat block for 3 minutes. Carefully remove tube (contents hot!) and vortex briefly 4-5 times for ~10 seconds each time. Repeat 100°C heat treatment and vortexing step again as above. Ask your instructor to inspect your sample to determine if it is completely lysed (solution should be appear clear blue with no chunky debris).

5. DNA SEQUENCE ANALYSIS: Using the DNA that you purified today in step (a), remove 12 ul of DAY 11 plasmid DNA and place it in a PCR striptube tube containing 3 ul of GFP DNA sequencing primer (5 uM) as directed by your instructor. DNA sequencing samples will be sent to Quintara Biosciences for sequence determination. We will analyze these results when the sequence becomes available.

DAY 12, Oct 17 MOLECULAR MODELING OF LEPA/EF-4 PROTEIN STRUCTURE; RESTRICTION DIGESTION OF DAY 11 AND DAY 2 PLASMID DNA. AGAROSE ELECTROPHORESIS

Purpose: To determine size of the GFP-positive plasmid

Reagents

Restriction enzyme reaction (uL)

ddH20

8.4

10X Buffer

1.6

pPEM109 plasmid DNA (from DAY 11)

5.0

XbaI enzyme

1.0

Total:

16

2. Incubate at 37 oC for 1 h. Add 4 uL of DNA sample loading dye to stop reaction.

DAY 13 Oct. 19 WESTERN BLOT ANALYSIS OF FUSION CANDIDATES

Purpose: To determine size of the GFP-positive plasmid and GFP-positive protein target

Background: To detect proteins from the GFP fusion candidate we will use gel electrophoresis to separate proteins based on size

Western Blotting is a method that can be used to detect the position of migration of a specific protein in a complex mixture of proteins that were separated by protein gel electrophoresis. When electrophoresis of a cellular lysate is followed by Coomassie Blue staining, a complicated pattern of bands is typically observed in the gel.

Protein Gel Electrophoresis:

Load 5 ul from +IPTG to the gel, load next to it 5 ul from –IPTG

· MW GFP 25,000

· MW LepA/EF-4 75,000

· MW LepA/GFP 100,000

After electrophoresis CAREFULLY place your gel onto a nitrocellulose membrane and assemble the blotting apparatus according to manufacturers instructions. Blot for 1 to 2 hours, and after blotting place the nitrocellulose filter into a 5% milk TBST solution for the blocking step.

DATA ANALYSIS

1) Does your Day 13 plasmid DNA agarose gel match the expected digestion pattern for a GFP insertion? Why or why not? Make sure to comment on both the number of bands, the expected and observed sizes of digested plasmid DNA, and reference your labeled data gel (including the ladder).

2) Discuss any potential sources of error in the experiment (even if not observed) and how that may have affected the outcome.