Reviving bacteria from 1 year old petri dish
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Mega [Andreas Stuermer]
For a workshop I gotta revive agrobacterium + GFP from the fridge.
I still have a one year old petri dish and a liquid culture, and a stab culture. Strangely, I got no colonies from the stab culture on a petri dish yet (1 day, but I know they grow very slow). Should have done fresh stabs half a year ago it seems. I always thought stab cultures are most durable?
I also tried putting the liquid culture on a petri dish, which also yielded no colonies after 1 day.
So finally I tried to pick tons of bacterial lawn/colonies from the petri dish, put them in liquid LB kan medium and it got a bit cloudy after one day.
Maybe the explanation - in liqiud medium the end-products are distributed more homogenously - so quicker growth but quicker stagnation/dying.
I was wondering, do the kanamycin degrading enzymes still work after one year?
Would you assume it's still the correct bacterium after one year - contamination wouldn't grow as quickly I believe. (I could do a transient GFP plant, but it takes 5 days and I would have to send it today)
Mega [Andreas Stuermer]
Claudio Tiecher
Keep in mind as long as there is one healthy cell it works! Try your luck!
The kanamycin degrading enzyme is probably broken/low activity after one year. That is not the problem becuase, as soon as the cell is in fresh medium, it just produces new one.
Mega [Andreas Stuermer]
Cathal Garvey
yes. But, even after a year, if they were stable at 4C you should be OK.
I've seen 4C in an agar stab recommended as the best way to store
plasmid-bearing cells, preferable over -20C and possibly even -80C, in
at least one reputable source. But that's a stab, whereas in an
overgrown petri dish things might be less stable.
Try your luck, but if it's kanamycin do try a recovery period I think.
On 28/01/15 13:25, Mega [Andreas Stuermer] wrote:
> But then you have to breed them for an hour in LB without kanamycin first to give them time to produce the enzyme?
>
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Andreas Stuermer
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Andreas Stuermer
Cathal Garvey
Or PCR?
On 28/01/15 14:05, Andreas Stuermer wrote:
> Well, we finally got tons of growth of the liquid medium directly plated
> onto a kanamycin agar plate. Without recovery.We put some colonies of
> this into LB kanamycin liquid medium and it grew cloudy.
>
> Unfortunatly we don't have time till the workshop to re-do this from the
> 1 year old agar plate -> LB for recovery -> LB kan for selection.
>
>
> Still, as it is kanamycin resistant I hope it is the correct bacterium
>
>
> On Wed, Jan 28, 2015 at 3:00 PM, Cathal Garvey
> <cathal...@cathalgarvey.me <mailto:cathal...@cathalgarvey.me>> wrote:
>
> I'd let them recover for 30m/1h in SOC or LB, then plate on
> kanamycin, yes. But, even after a year, if they were stable at 4C
> you should be OK.
>
> I've seen 4C in an agar stab recommended as the best way to store
> plasmid-bearing cells, preferable over -20C and possibly even -80C,
> in at least one reputable source. But that's a stab, whereas in an
> overgrown petri dish things might be less stable.
>
> Try your luck, but if it's kanamycin do try a recovery period I think.
>
> On 28/01/15 13:25, Mega [Andreas Stuermer] wrote:
>
> But then you have to breed them for an hour in LB without
> kanamycin first to give them time to produce the enzyme?
>
>
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Mega [Andreas Stuermer]
So I conclude it's very likely agro. We'll definitely see after transfecting the plant
Filip Hasecke
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Filip Hasecke
Maybe it's just dead cells that are dissolved in the medium that make it cloudy though... But there is no debris at the bottom of the tube.
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Mega [Andreas Stuermer]
Sunil Phani
Nearly forgot to reply... We got GFP plantlets, so agro seemingly survived.Unless there's another explanation for spontaneous green fluorescence where LB medium is applied :D
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Sunil Phani
OK, so we have a gene product of interest ("coagulation factor VIII, procoagulant component") that you would like to produce antibodies against. What next? Whether you are using phage display or immunizations, to raise antibodies you are going to need some of the protein product from that gene as a target. This collection of HOWTOs will walk you through the process of designing and producing good recombinant protein bait for your experiments.
This process falls into 4 steps:
1. recombinant protein design
2. clone gene
3. transfect cells
4. purify protein
This how-to will address #1, recombinant protein design.
Find your gene of interest
You’ll want to begin by finding the sequence for your gene of interest. The availability of genome projects and annotation databases makes it pretty easy to pull up sequences of genes from many species. The standard destination for recovering such sequences is NCBI.
By searching for your gene name in the NCBI welcome screen search fields, you should be able to find hits for “gene” and “protein.” Every gene has a lot of different names and accessions that can be used. NCBI does a reasonable job of interpreting common names, HGNC gene IDs and various accessions, but still make sure to read a bit of the page on the resulting hits to make sure you have arrived at the correct gene product. On the gene pages you will find a lot of information about this gene of interest, as well as the ability to extract the nucleotide and amino acid sequence of the gene. Copy these to your working project folder and a word document.
A common error in antigen design is the assumption that at this point you are done. You are not done! A raw cDNA sequence cannot be purified, will usually not be secreted, may not be very soluble, and might be tethered to cell membranes. Before you are ready to produce your recombinant protein, you are going to need to engineer it to be suitable for mammalian secreted expression and his-tag purification.
At the nucleotide level, genes can be separated into a series of discrete modular components: a promoter, a series of exons and introns, and a termination stop codon. In almost all cases you don’t need the introns: they will just cost more to synthesize and potentially complicate products produced. The easiest way to get rid of them is to pick the cDNA version of the gene. This cDNA version has already had the introns spliced out: it should translate into the amino acid encoding of the gene with no stop codons. Note that genes can have multiple splice variants that combine subsets of the available exons: make sure to pick the version that contains the protein folding domain(s) you want to target (see below).
At the amino acid level, mammalian genes can be also be separated into a series of modular components: the leader sequence that influences expression and localization, a series of protein folding domains that produce the resulting protein fold and gene function(s), and potentially a set of one or more transmembrane domains that tether your protein to cell membranes. These modular units often, but not always, coincide with exons. Most genes have been carefully annotated to reveal the locations of these different domains.
To identify these regions, I recommend using the UniPROT human curated database:
Search UniPROT with your protein of interest and you should find a number of potential hits. Chose the hit that is in the species you care about (i.e. human). If you have a choice, choose the hit that has a star next to it: this indicates that this gene has undergone human curation by experts. Double check that the aliases for the gene at the page you arrive at make sense (i.e. that you haven’t arrived at a different gene that just shares this same protein’s name).
The page will contain a region that highlights where the leader sequence, domains and transmembrane regions are for your sequence. It will also indicate allelic variation, splice site variants, and a series of studies addressing the function, analysis, and maybe even expression of this protein.
To supplement the data available on UniPROT, you should also pull up any available crystal structures of your protein, or a closely related homolog. You can do this from the database PDB.
http://www.rcsb.org/pdb/home/home.do
PDB can be searched by gene name, but it can also be searched by amino acid sequences. It is best to search using the amino acid sequence of the protein domains that you would like to target, as this will help recover hits of proteins that were named differently (PDB isn’t very smart about figuring out protein aliases) and will also recover homologous proteins that might not be your exact protein, but represent a distant cousin that likely adopts the same fold.
In order to visualize the resulting protein I recommend using UCSF Chimera:
https://www.cgl.ucsf.edu/chimera/
Install Chimera, and then import PDB structures using
File -> Fetch by ID
Enter the PDB ID of the structure you had looked up in PDB, and it will become available in Chimera. Spend a little time comparing the protein domains described in UniPROT to their locations on the physical structure. This will be your guide and sanitry control when making good decisions about what truly constitutes the edge of a folding domain.
OK, so now you have the NCBI documents, the UniPROT documents, and the PDB crystal structure documents for your gene of interest open. Let’s begin the engineering:
Promoter
The native promoter influences how much your protein expresses, under what tissues and under what circumstances. Since our goal is to produce as much protein as possible constantly, we are going to skip the native promoter and instead use the one in our mammalian expression vector. These are usually derived from viruses like CMV or other hyper-active genes that produce a tremendous amount of protein constantly.
Leader sequence
The leader sequence (leader peptide, signal sequence) is a set of 5-30 residues at the N-terminus of your protein that begin with an M (Methionine) and are usually notably hydrophobic. The leader sequence can influence the expression level and cellular localization of your resulting protein. Native leader sequences can cause all sorts of annoying problems, so you will want to remove it and replace it with our vector’s default leader sequence. This sequence is especially designed to traffic the proteins into a secretory pathway so that your protein ends up secreted outside of the mammalian expression cells into the supernatant. This avoids a concentration build-up in the cells, and makes it much easier to recover your proteins (you can just spin down the cells and purify your protein from the supernatant). Make sure not to accidentally include the native leader sequence in the recombinant protein.
Transmembrane domains
Transmembrane domains weave proteins through membranes so that they can carry out various roles in various cellular compartments. This weaving is very systematic: a receptor with two domains separated by a transmembrane domain will always be produced so that on domain (for example, the receptor domain) is always found outside of the cell, while the second domain (for example, a kinase domain) is always found inside the cell. They are important for the cell, but terrible for protein expression: your protein will get stuck in membranes and not secreted, or horribly aggregate due to the hydrophobic sticky peptide that each protein now carries with it. Although there are ways to express and purify membrane-bound proteins, we aren’t going to be addressing them here. Instead, we will analyze our protein to identify only the extracellular domains that we would like to express. Antibodies only easily access the outside of cells, so we don’t really care about intracellular domains. If we find any transmembrane domains, then we will want to see which domains of our protein are found in the extracellular space. For a single-pass transmembrane protein (i.e. a receptor) we will want to identify the extracellular portion and the exact region that the transmembrane domain begins. You will then want to produce a soluble extracellular-only version of the receptor. This is where UniPROT and PDB will be very helpful. In general, you will want to include as few transmembrane residues as possible (as they are hydrophobic and sticky), but not so few that the fold becomes less stable (UniPROT sometimes is only aggressive on the declaration of the end of the domains). Cross-check UniPROT by looking at the structure and making sure that you are cropping the transmembrane domain at a point where it is no longer forming a large number of contacts with the rest of the nearby fold.
This is a bit of an art, but a few things can help you be successful here. First, many receptors produce soluble-only versions of their receptors. This is nature telling you where the correct boundaries for a soluble receptor variant lie. Second, if you see a crystal structure of a protein, it means that material had to have been successfully expressed in bulk. See what they chose as their residue cutoffs (but make sure to read their methods to make sure they didn’t do anything particularly complicated to express their protein). Third, almost all proteins that you will be working on will have been worked on by other groups already. Spend a little time on google scholar to search for your gene of interest along with “soluble expression” or “mammalian expression” to see if this problem has already been solved for you. Finally, be aware that some allelic variants and splice site variants have expression and folding problems. It’s worth scanning the UniPROT contents briefly to make sure you aren’t using one of these problem sequences.
His-tag purification
OK, you’ve swapped out the promoter, swapped out the leader sequence, picked the extracellular exons you care about, and are ready to go. You sequence should now express nicely and be secreted out into the supernatant of mammalian expression culture. But wait! Before you can order up your sequence, you’ll need a way to purify your resulting protein. The way this is typically done is by attaching a protein purification tag to the end of your sequence. A number of tags are available: we are going to us His-tag purification. The concept is pretty simple: at the C-terminal end of your protein you are going to attach a sequence that encodes 6 histidines in a row (i.e. HHHHHH). The amino acid Histidine is pretty awesome in that it changes from being polar to charged depending on the pH of the surrounding solution (it protonates at around pH 6). The his-tag binds nickel beads at high pH but not low pH. We can use this to purify and then elute (release) our protein by running the mammalian cell supernatant over nickel beads, washing off all other proteins, and then eluting our target protein by lowering the pH and/or applying compounds that compete with His for nickel binding.
A common mistake is to just attach the his-tag at the C-terminal end of the protein. While this sometimes works, it is also sometimes the case that the shape of the protein will cause the next few his-residues to be in a pocket that isn’t easily accessible to the his-tag. This is called steric hindrance (getting in the way). You can take a look at your PDB structure to see how much of an issue this is likely to be, but to ensure that your his-tag is easily available to bind nickel, the easiest solution is to add a small tether at the C-terminal end before you’re his-tag starts. The classic tether used by protein engineers is the serine-glycine linker: GGGSG. This provides a flexible, slightly polar linker that will help get the his-tag away from the main protein and out into solution.
Stop codon
You will need a stop codon at the end of your protein. Be safe – add multiple stop codons in different reading frames to defend yourself fro read-through. Your vector may already have this built in.
Expression encoding
You should have the native amino acid sequences for the extracellular domain as well as the nucleotide sequence that encodes this same region. You can encode the gs-linker and the his-tag any way you like, but in general it’s better to use different codons so as not to run up a long repeat of the same codon. Note that some codons perform better in mammalian expression than others: pick high functioning codons.
Your final protein should thus look like
[extracellular domain]-[GGGSG]-[HHHHHH]-[multi-stop]
At this point you will want to assemble the nucleotide version of your sequence, check your work to make sure it translates into the amino acid sequence you were expecting. You are almost done.
Vector formatting
Before you send off your sequence to by synthesized, you are going to need to add in adapters on the 5’ and 3’ end of your sequence. These adapters are going to include 40bp of sequence that overlaps the vector on each side of the region where you want to insert your sequence. This overlap contains the restriction sites that will be used during the cloning, and are long enough that you could also use the Gibson reaction to skip the cloning step if you desire. Not that synthesis companies like the ends of synthesized products to be A/T rich, so end the sequence at an A/T rich region of the vector.
Once you have added these regions to the ends of your sequence, there is one final, critical check. Search through your sequence to make sure that the restriction sites that you are going to use only appear in the expected locations, and not anywhere internal to the sequence. If they do, change codons in that region until the restriction sites are disrupted. If you don’t do this, your gene will get chopped up right when you begin preparing it for insertion into the vector (note: Gibson is a backup plan here, but there’s no reason not to save yourself some pain).
thanks you in advance for helping me to get my data verified