Introduction to the project:

(Note: Our commercial source of DNA in this project- plasmid vectors and expression clones- is Origene.)

Your team will choose one of several available eukaryotic genes (in plasmids) to work with for the semester-long project, each of which encodes a different protein involved in both normal and pathological human cell physiology. This gene is in the form of its coding sequence, or “ORF” (Open Reading Frame, minus introns, beginning with its start codon ATG) engineered into a plasmid “vector” to create an “expression clone” (see Figure 1). This expression clone is designed to enable transcription and translation of the gene of interest (GOI) in the appropriate mammalian host cells. (In this context, we are using the terms, “ORF” and “GOI” interchangeably).

 

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Figure 1: Example of an ‘expression clone’ containing an “ORF” (gene of interest) upstream of (5’ of) and in-frame with the “turbo Green Fluorescent Protein” (tGFP) gene. During transcription of the ORF in mammalian cells, RNA polymerase will ‘read-through’ and transcribe the tGFP gene as well, generating mRNA that is a fusion of the two genes. Translation of this ‘combination’, hybrid mRNA will generate a fusion protein consisting of the protein product of the gene of interest with a C-terminal fluorescent tGFP ‘tag’.

Visit the following link for more information about Green Fluorescent Protein and its versatility.

Referring to the first steps of the flow chart: you will be given a very small quantity (in the ‘ng’ range) of a plasmid containing your gene of interest; in order to work with this construct (clone) and generate any meaningful data, you will need much more plasmid DNA than what you were given. Transformation of plasmid DNA into competent bacteria (see below), followed by amplification in broth culture and plasmid purification, will accomplish this. After purification (in the following weeks), you will need to verify that your plasmid clone is the ‘correct’ one that you expected (as opposed to a different gene or the “empty” vector plasmid) and also to verify that it is not damaged/rearranged in the process of transformation and amplification in bacteria. Two diagnostic steps, PCR and restriction digestion, will be carried out to provide confirmation of the identity and integrity of your clone. The plasmid DNA will then be transfected into mammalian cells (later in the semester), followed by testing of cellular functions in the presence of the fusion protein.

Plasmid clones available: These links take you to Origene’s webpages, where you can find information and references on the genes, protein products, and cloning schemes.

1. RASSF1A_tGFP[1] (“Gene A”) RG213525
2. Fas_tGFP (“Gene F”) RG204520
3. b6 Integrin_tGFP (“Gene B6”) RG217387

In the following 2 clones, the “GFP” tag is another version of GFP termed “monomeric” GFP, or “mGFP”. For our purposes, ‘mGFP’ and turbo GFP (‘tGFP’) are functionally equivalent, and we only need to keep in mind that they are different proteins and therefore have different gene sequences. The clones below were not commercially available from Origene directly in their mGFP vectors; #4 was a ‘custom subclone’ given to us as a sample from Origene, and #5 was subcloned by SAC, from an “Entry vector”. Therefore, we don’t have a ‘single link’ to the clones as we do for the first three:

4. TNFR1A_mGFP (“Gene T”) RG204008 ORF subcloned into PS100040
5. MT1-MMP_mGFP (“Gene M”) RC208917 ORF subcloned into PS100040

 

Transformation of “Competent” Bacteria with Plasmid DNA

Background of transformation procedure:

Please refer to the “Cloning DNA” sections of your e-textbook, for useful background information on some of the common procedures used in cloning. Also, we recommend that you view a couple of helpful animations on the process of transformation:

http://www.dnalc.org/ddnalc/resources/transformation1.html

http://www.dnalc.org/resources/animations/transformation2.html

‘Transformation’ in general refers to the uptake of foreign DNA into host cells (‘foreign’ DNA could be from the same or a different species relative to the host). For our purposes today, it describes the process of introducing plasmid DNA (with engineered genes of interest) into host bacterial cells that have been prepared (usually with CaCl₂ solution) to uptake the DNA, making them ‘competent’. Without this prior treatment, uptake of foreign DNA by bacteria would be an extremely rare event. Even with the treatment, it is still a relatively inefficient process, requiring selection of the smaller number of ‘transformants’ from a larger background of untransformed cells. Antibiotic resistance is a common selection mechanism, which is why plasmids that are engineered for cloning purposes typically have one or more genes that will confer antibiotic resistance in their bacterial hosts. (Think of the LB agar plates that we just poured, containing the antibiotic Ampicillin.)

Transformation of bacteria with expression clones

Overview:

You will introduce your ‘expression clone’ (with your “gene of interest”) into competent bacteria, followed by overnight culture on LB-agar/ampicillin plates to obtain colonies of transformed cells. (Each colony is a clone, derived from a single bacterial cell.) This will be followed by colony harvest and plasmid purification.

Reagents, etc.:

• Competent DH5α bacterial cells
• LB agar-ampicillin plates (3 per group)
• LB agar plates (with no amp) (1 per group)
• SOC[2] broth (without ampicillin)
• TE buffer (10 mM Tris-1 mM EDTA, pH 8.0) (prepared by each team)
• Bacterial cell spreaders, autoclaved (re-useable)

Lab Instrumentation:

• Water baths at 42°C
• Bacterial incubator/shaker at 37°C (408)

Note: At warmer temperatures (room temperature or 37⁰C), competent bacteria very quickly repair the damage done to their cell walls and can lose their “competence” for taking up DNA. Therefore, they must be kept on ice after thawing in order to minimize the loss of competency.

 Bacterial Transformation:

1. Choose your team’s gene/plasmid from those that are available; plasmid DNA concentration = 1 ng/ul.

Competent DH5α cells from the -80ºC freezer will be thawed on ice for ~5 minutes by the NINJAs. Cells will be made available for you when you are ready.

2. Obtain and label 2 sterile microcentrifuge tubes: one is “negative” and one is “Gene _” (A, F, T, M, etc); include your team name (or a logo) on the tubes, so that you will be able to recognize them among the others. Place these tubes on ice to pre-chill them.

3. With gentle pipetting, add 30 ul competent DH5acells to each Note: This is one of those rare instances in which you MUST NOT pre-mix the contents of the tube of cells before removing your sample; competent cells are fragile and must not be vortexed or vigorously pipetted.

4. Add 2 µl of the plasmid DNA (2 ng) to the cells in the tube labeled “Gene _”; mix gently by swirling the pipette tip around in the mixture (“stirring”). Similarly, add 2 µl of TE buffer to the cells in the tube labeled “negative” and mix. DO NOT mix by pipetting up and down. DO NOT ‘pulse-spin’ in the microcentrifuge.
5. Incubate on ice for 30 minutes. This step allows the cells and the DNA to complex together in the presence of the CaCl₂ solution that the cells are in.

6. Heat-shock cells:
   1. Place tubes in 42ºC water bath for 45 seconds.
   2. Immediately return the tubes to ice for 2 minutes. (Note: these changes from ice to 42⁰C and then back to ice must take place very quickly. Bring your ice bucket and timer to the back of the room and stay near the water bath for these quick temperature changes.)

7. Add 450 µl of room temperature SOC medium (without ampicillin)[3] to each tube of cells.

8. Cap and place the tubes at a 45⁰ angle in the shaking (~225 rpm) bacterial incubator, room 408, which is set to 37⁰ Incubate for 30 minutes.

During the incubation, label the bottom edges (not the center!) of 3 LB-agar/ampicillin plates with your team name, the date, and the following:

Negative – 40 µl

Gene ‘_’- 20 µl

Gene ‘_’- 40 µl

Also, label a single LB agar plate (with no amp) with your team name, date, and:   

Gene ‘_’- 40 µl

9. Remove the bacteria from the 37⁰C incubator upon completion of the recovery phase. (The following step will be demonstrated by Instructor.): Prior to pipetting, briefly mix each tube by gently inverting a few times. Pipette the cell suspensions directly over the center of your appropriately labeled LB plates as indicated below; spread evenly with a sterile yellow spreader immediately afterwards. Use a different spreader for each sample! After use, place each spreaders in a beaker of 70% ethanol.

 

• Negative control (TE buffer only): 40 l onto LB/amp
• Gene- 20 µl onto LB/amp
• Gene- 40 µl onto LB/amp
• Gene- 40 µl onto LB (no ampicillin)

After spreading the cells, leave the dishes covered and upright for a few minutes. Then invert the plates and incubate (inverted) overnight at 37ºC.

10. Instructor will remove the plates from the incubator the following morning, wrap the stack in parafilm and store at 4ºC until the next step.

Points to address in your lab notebook after observing your colonies

1. Clearly identify the gene you are working with, and briefly describe its normal function in cells.
2. What do you expect to see on the LB/amp plate that was spread with ‘negative control’ cells (‘transformed’ with only TE buffer)? Why did we include this control? What are we testing? (Could be more than one answer)
3. What do you expect to see on the LB plate (no antibiotic) that was spread with the ‘Gene X’ -transformed cells? Why?
4. What purpose does the ampicillin serve? (Make sure you fully understand this.)

Importantly, prior to observing your plates after colony growth, try and anticipate what you should see on each plate. If you see the ‘unexpected’, what could it mean? (Trouble-shooting is very important in laboratory work!)

[1] “tGFP” refers to Origene’s “turbo GFP”, a version of GFP that needs to dimerize (2 units combine) for fluorescence. “mGFP” is Origene’s “monomeric GFP”, which doesn’t dimerize.

[2] SOC = Super Optimal broth with Catabolite repression; essentially it is Super Optimal Broth (a rich bacterial growth media) with added glucose. For the purpose of ‘recovery’ of DH5a cells after transformation, it appears to work better (yield more transformants) than LB broth.

[3] At this point, we just want the newly transformed, fragile cells to “recover” and start replicating, and to produce the protein product of the amp resistance gene (b-lactamase). Secreted b-lactamase will cleave and inactivate ampicillin, which will allow the desired transformants to replicate on LB-agar/ampicillin in the next step.