2012 DIARY

ISIS Report 19/03/12

Genetic Engineering Spider Silk

Recombinant spider silk can be produced safely and effectively in contained facilities from genetically engineered bacteria and silk worms, provided containment is strictly implemented Dr. Mae-Wan Ho

A fully illustrated and referenced version of this article is posted on ISIS here

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Uses of spider silk

The potential applications of spider silk are legion on account of its unique combination of mechanical properties, not least the shimmering beauty of the natural fibre (see [1] In Praise of Spiders and Spider Silk, SiS 54), which is stronger than steel yet flexible and extensible, making it the toughest material available, hence the bulletproof vest of the headlines (see [2] Unspinning the Web of the Spider Goat, SiS 54). Apart from providing new fibres for textiles for a multitude of purposes, spider silk is ideal for use as surgical suture, as scaffolds for bone and skin in culture for tissue replacement, as drug delivery capsules, for solid states devices, biosensing films [3], parachute cords, and composite materials in aircrafts [4].

But as spiders are very difficult to farm, a great deal of effort has gone into producing spider silk by genetic engineering.

There are major challenges in producing spider silk through genetic engineering. First, the repetitive modules of the spider silk gene [1] tends to become truncated in the transgenic host through recombination between repeated modules of the same gene. Second, the high content of glycine (43-45 %) puts an unusual burden on the translation machinery of the host cell [3]. And even after the perfect silk protein is produced, the biggest hurdle of all is to spin a fibre that’s as good as the one the spider spins naturally with the greatest of ease.

The repetitive nature of the protein core, nevertheless, offers opportunities of creating synthetic genes that could modify and tailor the mechanical properties of the silk for specific applications.

The longest spider silk molecules engineered in E. coli and produced at high levels

Among the most successful attempts so far is the production of spider silk in genetically modified E. coli bacteria.

A group of researchers led by San Yup Lee at Korea Advanced Institute of Science and Technology in Daejeon have succeeded in producing the largest spider silk proteins in E. coli. A record-setting 284.9 kDa recombinant silk protein of Nephila clavipes (a Golden Orb spider species) was produced and spun into a fibre displaying mechanical properties ‘comparable’ to those of the native silk. The glycine-rich protein was favourably expressed in a metabolically engineered strain with an enhanced pool of glycyl-tRNA, the transfer RNA of glycine charged with the amino acid and ready to be added to the growing spider silk protein chain.  

The large protein produced was important, as smaller proteins gave inferior silk fibres, and the enrichment of glycyl-tRNA boosted the production of silk proteins to high levels.

The researchers used synthetic versions of the major ampullate spidroin 1 (MaSp1) [1] containing different number of repeat modules from 32 to 96, with predicted molecular weights of 100.7 to 284.9 kDa, the largest being similar in molecular weight to the natural MaSp2 found in the spider’s silk gland.

To find out how silk gene expression affects the host proteins, the researchers performed proteomic analysis, which gives a profile of the level of expression of all the proteins in the cell. Many stress response proteins were upregulated. Importantly, the enzyme that synthesizes glycine - serine hydroxymethyltransferase (GlyA) - and the b-subunit (GlyS) of the enzyme that synthesizes glycyl-tRNA were both upregulated, suggesting an increased demand for glycyl-RNA in expressing the glycine-rich protein.

An increase in the gene dosage of glycyl-tRNA synthase did not enhance silk production. So it was decided to increase the glycyl-tRNA pool by overexpressing the genes encoding the tRNAs in a plasmid. This resulted in higher expression of the four larger spider silk proteins (48-96-mer), and in all instances, enhanced cell growth by 30-50 %. The expression levels of the top two largest proteins (80-96-mer) were further increased by duplicating the glycyl-tRNA expression cassette. In addition, the glycine biosynthetic enzyme, GlyA was over-expressed, which further boosted the levels of the three largest proteins. The two modifications acted synergistically; together, they allowed 10 to 35-fold higher production of the three largest spider silk proteins.

The bacterial strains could be grown to high cell densities of >42 g/l, with maximum silk protein concentrations estimated in the range of 0.5 to 2.7 g/l (minimum estimates).

The cells were collected from 1 litre of culture, and lysed (burst open). Acid was added to the suspension, which got rid of most of the contaminating cell proteins, as silk proteins are highly positively charged, and hence acid-soluble. Fractional ammonium sulphate precipitation further enriched and purified the silk protein. Further steps resulted in  1.2 g of the silk protein with a purity of about 90 %.

Spun fibres comparable with natural fibres

Fibres were spun from all the different proteins, including a 16-mer 54.6 kDa protein, under the same spinning and drawing conditions after dissolving in hexafluoroisopropanol at a concentration of 20 % (w/v).

The mechanical properties of a fibre [1] are best described by the shape of a stress-strain curve when the fibre is stretched. The maximum height of the stress-strain curve is called tenacity, the amount of stress that a fibre can take before being torn apart. The furthest horizontal extent of the stress-strain curve is breaking strain, representing the extensibility of the fibre. The ratio of stress to strain is Young’s modulus, which is derived from the slope of the curve as a measure of the fibre’s stiffness. The 96-mer fibre exhibited a tenacity of 508 + 108 MPa and elongation of 15 + 5%, which are comparable to the values reported for native N. clavipes dragline silk (740–1 200 MPa; 18–27%). Notably, Young’s modulus of the 96-mer fibre was 21 + 4 GPa, twice that of the native dragline silk (11–14 GPa). Previously, a 60 kDa recombinant dragline silk protein of the spider Araneus diadematus was spun into a fibre with average tenacity ~ 260 MPa,  4.2-fold below that of the native silk at 1 100 MPa, possibly due to the small size of the recombinant protein. The tenacity of the 96-mer fibre (508 + 108 MPa) is the highest ever reported for recombinant spider silk proteins (see Figure 1).

Figure 1   Analysis of fibres spun from recombinant silk proteins; a, Typical stress strain cruves of 16-mer (blue), 32-mer (red), 64-mer (green) and 96-mer (purple) fibres, from Xia et al, 2011 [4]

Scanning electron microscopy analysis of fibre surface revealed fibrillar structure in all the fibres, as for native spider dragline silk. Analysis of fractured fibres showed many irregular voids in the shorter fibres, whereas those defects were absent in the 64- and 96-mer fibres, and suggests why longer silk proteins make stronger fibres (Figure 2).

Figure 2  Scanning electron micrographs of the fibres spun from (left to right) 16-mer, 32-mer, 64-mer and 96-mer proteins; top surface of fibres, middle, and bottom, fractured surface, scale bar top and middle 10 mm, bottom 1 mm, from Xia et al , 2011 [4]

The E. coli system has the great advantage that the bacterial genome is well-characterized and understood, and can be manipulated quite precisely. It holds considerable promise for producing good quality spider silk efficiently and safely under strictly contained conditions. One disadvantage is that it cannot produce the silk fibre in a ready-spun form.

Hybrid spider silk from silkworms

The silkworm Bombyx mori is another promising host for spider silk production. A major limitation for producing good quality spider silk is the spinning process that makes fibres from the silk proteins. Transgenic silkworms can potentially produce recombinant spider silk fibres ready-spun, provided that the spider silk protein is targeted to the worm’s silk gland with tissue-specific promoter.

A team of researchers led by Donald Jarvis at the University of Wyoming, Laramie, in the United States used the piggyBac insect vectors to transform silkworms with a synthetic spider silk gene containing 14 repeats of a module containing (A)8.(GPGGA)8, where A stands for alanine, G, for glycine, P for proline. The repeats were joined to the C- and N- termini peptides of the silkworm fibroin heavy chain gene, and placed under the control of the fibroin heavy chain promoter and enhancer sequences. The construct was designed to produce worm-spider hybrid silk that can be isolated from the worm’s cocoon. In another version of the vector, the spider silk sequence was also joined to a green fluorescent protein gene to aid in identifying transgenic silks and worms.

Transgenic silkworms were produced, and those with the green fluorescent protein readily identifiable by visual inspection of the cocoons as well as the silk glands. The chimeric silkworm/spider silk was analysed and determined to be about 2 to 5 % spider silk.  The hybrid silk proteins were about 100 to 130 k Da.

Despite the low levels of spider silk protein incorporated into the chimeric fibres, they were significantly tougher than the silkworm fibres, and as tough as the native dragline silk, even though not as strong.

Like the E. coli system, the transgenic silkworms can be kept under strictly contained conditions, and holds much promise for farming spider silk or hybrid spider silkworm silk with the requisite properties. It is important to note that the piggyBac insect vector is a transposon, and has a tendency to remobilize and spread by horizontal gene transfer (see [6, 7] Regulation of Transgenic Insects Highly Inadequate & Unsafe, and Transgenic Mosquitoes Not a Solution, SiS 54). That is why transgenic silkworms created using this method must be reared under strictly contained conditions.

To conclude

Recombinant spider silk can be produced safely and effectively under strictly confined conditions in transgenic E. coli and transgenic silkworms. There is no reason to continue with production in transgenic goats, a system that has remained characterized, and is neither ethical nor safe [2].

GRAPHENE
by Nick Hodge | Friday, March 23rd, 2012
(PLEASE OBSERVE WHILE READING THIS THAT THIS MATERIAL CAN REPLACE OIL PRODUCED MATERIALS - plastics etc. FOR MODERN FACILITIES AND ENHANCE THEM CONSIDERABLY. J.B.Editor)

1. Graphene made from weakly bonded layers of carbon - graphite.
2. It is composed of carbon atoms arranged in tightly bound hexagons just one atom thick
3. 3,000,000 sheets of grapgene would be 1mm thick
4. The bond structure of graphite was first theorised by P.R.Wallace in 1947, though for it to exist was regarded as impossible.
5. In 2004 teams including Andre Geim and Konstantin Novoselov demonstrated that single layers can be isolated resuling in the award of the Nobel Prize for physics in 2010.
6. It is a good thermal and electric conductor and can be used to develop semi-conductor circuits and computer parts.

I've been tight-lipped for a while, but it's finally time to show you what the BBC calls a “Miracle Material.”
Its actual name is graphene, given by the two scientists who won a Nobel Prize for their discovering it.
And it's going to change the world...
“Graphene doesn't just have one application,” says Andre Geim, who made the find along with Konstantin Novoselov.
"It is not even one material. It is a huge range of materials. A good comparison would be to how plastics are used."
I'd say plastics is a conservative comparison.
Let me show you what hundreds of researchers, companies, and governments are already doing with the strongest, thinnest, most conductive material ever discovered.
The BBC says: “It could spell the end for silicon and change the future of computers and other devices forever.”
The Daily Mail says: “A graphene credit card could store as much information as today’s computers,” and that “graphene will lead to gadgets that make the iPhone and Kindle seem like toys from the age of steam trains.”
But it won't just revolutionize electronics...
Graphene is also being used for energy, defense, and medicine applications.
Engineers at Northwestern University have a made a graphene electrode that allows lithium-ion batteries to store 10 times as much power and charge 10 times faster.
MIT Engineering Professor Jeffrey Grossman believes solar cells made from graphene could produce 10,000 times more energy from a given amount of carbon than fossil fuels.
And CNBC reports it could expand the current domestic oil boom because “tiny sensors coated with the wonder-material graphene and powered by flowing water could expedite the discovery of oil and natural gas reserves.”
It may sound too good to be true, but I assure you it isn't.
Take it from co-Nobel recipient Konstantin Novoselov:
I don’t think it has been over-hyped. It has attracted a lot of attention because it is so simple — it is the thinnest possible matter — and yet it has so many unique properties. There are hundreds of properties which are unique or superior to other materials. Because it is only one atom thick it is quite transparent — not many materials that can conduct electricity which are transparent.

Experiments have shown that it is incredibly strong .

(ScienceDaily (Mar. 21, 2012) — The Graphene Research Group at Toyohashi University of Technology have synthesized graphene by reducing graphene oxide using microorganisms extracted from a local river.)
ScienceDaily (Feb. 5, 2010) — In a just-published paper in the magazine Science, IBM researchers demonstrated a radio-frequency graphene transistor with the highest cut-off frequency achieved so far for any graphene device -- 100 billion cycles/second (100 GigaHertz).
Nanotechnology Circuits for Wireless Devices: First Wafer-Scale Graphene Integrated Circuit Smaller Than a Pinhead (June 11, 2011) — Scientists have achieved a milestone in creating a building block for the future of wireless devices. Researchers have announced the first integrated circuit fabricated from wafer-size graphene,
Key Milestone Reached On Road to Graphene-Based Electronic Devices (Feb. 1, 2010) — Researchers have produced 100mm diameter graphene wafers, a key milestone in the development of graphene for next generation high frequency electronic devices.

nuclear reports
Fukushima:Very high radiation, little water in Japan reactor

By MARI YAMAGUCHI, Associated Press – 13 hours ago

TOKYO (AP) — One of Japan's crippled nuclear reactors still has fatally high radiation levels and hardly any water to cool it, according to an internal examination that renews doubts about the plant's stability.

A tool equipped with a tiny video camera, a thermometer, a dosimeter and a water gauge was used to assess damage inside the No. 2 reactor's containment chamber for the second time since the tsunami swept into the Fukushima Dai-ichi plant a year ago.

The data collected Tuesday showed the damage from the disaster was so severe, the plant operator will have to develop special equipment and technology to tolerate the harsh environment and decommission the plant, a process expected to last decades.

The other two reactors that had meltdowns could be in even worse shape. The No. 2 reactor is the only one officials have been able to closely examine so far.

Tuesday's examination with an industrial endoscope detected radiation levels up to 10 times the fatal dose inside the chamber. Plant officials previously said more than half of the melted fuel has breached the core and dropped to the floor of the primary containment vessel, some of it splashing against the wall or the floor.

Particles from melted fuel have probably sent radiation levels up to a dangerously high 70 sieverts per hour inside the container, said Junichi Matsumoto, spokesman for Tokyo Electric Power Co. The figure far exceeds the highest level previously detected, 10 sieverts per hour, which was detected around an exhaust duct shared by No. 1 and 2 units last year.

"It's extremely high," he said, adding that an endoscope would last only 14 hours in those conditions. "We have to develop equipment that can tolerate high radiation" when locating and removing melted fuel during the decommissioning.

The probe also found that the containment vessel — a beaker-shaped container enclosing the core — had cooling water up to only 60 centimeters (2 feet) from the bottom, far below the 10 meters (yards) estimated when the government declared the plant stable in December. The plant is continuing to pump water into the reactor.

Video footage taken by the probe showed the water inside was clear but contained dark yellow sediments, believed to be fragments of rust, paint that had been peeled off or dust.

A probe done in January failed to find the water surface and provided only images showing steam, unidentified parts and rusty metal surfaces scarred by exposure to radiation, heat and humidity. Finding the water level was important to help locate damaged areas where radioactive water is escaping.

Matsumoto said that the actual water level inside the chamber was way off the estimate, which had used data that turned out to be unreliable. But the results don't affect the plant's "cold shutdown status" because the water temperature was about 50 degrees Celsius (122 Fahrenheit), indicating the melted fuel is cooled.

Three Dai-ichi reactors had meltdowns, but the No. 2 reactor is the only one that has been examined because radiation levels inside the reactor building are relatively low and its container is designed with a convenient slot to send in the endoscope.

The exact conditions of the other two reactors, where hydrogen explosions damaged their buildings, are still unknown. Simulations have indicated that more fuel inside No. 1 has breached the core than the other two, but radiation at No. 3 remains the highest.

The high radiation levels inside the No. 2 reactor's chamber mean it's inaccessible to the workers, but parts of the reactor building are accessible for a few minutes at a time — with the workers wearing full protection.

Last year's massive earthquake and a tsunami set off the worst nuclear accident since Chernobyl, sending three reactor cores to melt and causing massive radiation leaks. The government said in December that the reactors are safely cooled and the plant has stabilized, while experts have questioned its vulnerability.

During a recent visit by a group of journalists including The Associated Press, the head of the plant said it remains vulnerable to strong aftershocks and tsunami, and that containing contaminated water and radiation is a challenge. Radioactive water had leaked into the ocean several times already.

Workers found a fresh leak of 120 tons from a water treatment unit this week from one of its hoses, with estimated 80 liters (20 gallons) escaping into the ocean, Matsumoto said. Officials are still investigating its impact.

Fukushima's accident has instilled public distrust and concerns about nuclear safety, making it difficult for the government to start up reactors even after regular safety checks. All but one of Japan's 54 reactors are now offline, with the last one scheduled to stop in early May.

Copyright © 2012 The Associated Press. All rights reserved.
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SanOffre Nuclear Power Station U.S.A.:Update to the letter below:28.3.2012
Dear Readers,
Last week former U.S. Vice President Dick Cheney was the recipient of a "new" heart.
Long live the automobile industry, where most of the "organ donors" in America come from!
Ugh, what's wrong with THAT sentence? I mean besides the obvious: That someone more deserving than Dick Cheney might have deserved that heart? If you can figure it out, perhaps you can understand what's wrong with nuclear power. Yes, it's really that simple! Nuclear power kills. There might be some benefits, but along the way, it kills. There are vastly safer ways to either conserve or generate electricity without increasing global warming. We don't need nukes. Would it be argued that we need car accidents just to be sure we have organ donors?

Vice President Cheney's old heart was no good (what a surprise!): He had quadruple bypass surgery 24 years ago, then two angioplasties, and a heart monitoring device put in (later removed). There wasn't much left to do but replace the thing! He reportedly was on the "waiting list" for a suitable donor organ for the past 20 months.

San Onofre Nuclear (Waste) Generating Station in Southern California is falling apart, and so they keep putting new parts in. Last time, it was new turbine blades (for "greater efficiency" they said) and a new reactor pressure vessel head (because the old one was rotting, decaying, corroding, embrittling, rusting -- you get the idea).

The time before that, a little over a year ago, it was four new steam generators, two per reactor. The time before that it was something else, and something else the time before that. Billions of dollars worth of parts -- but billions more dollars worth of parts are NOT being replaced UNLESS they fail! And the new parts aren't working, anyway.

The ratepayers are paying for everything, of course. It's always the ratepayer who pays, so the utility's investors can make money. The California Public Utilities Commission ensures that electricity rates will be sufficient so that the utility will make money. How much? Enough to keep the utility happy.

But Southern California Edison is not so happy right now, because their #1 cash cow isn't giving any milk. SanO has been completely shut down for nearly two months and isn't likely to reopen any time soon. So the utility is losing about a million dollars a day per reactor. They could get most of the money some other way, because people NEED electricity and they can supply it, or at least help us exchange it with each other. But nuclear reactors, once operating, are relatively cheap to maintain -- as long as the ratepayer covers most extra costs. And as long as someone (John Q. Public) takes the waste away eventually, or at least promises to (the federal government promised to, but hasn't done so). And as long as nothing goes wrong. Then they can be very, very expensive, as they learned in Japan and the Ukraine, and as we hope NOT to learn firsthand here.

Your main pump -- your heart -- can fail in many ways. The muscle of the pump can fail to get the proper bioelectrical signal that tells it to beat. If this potential problem is noticed in time, a pacemaker can be implanted so that an artificially-produced electrical signal can be applied to the heart.

At the Brown's Ferry reactor in Tennessee in 1975, a careless worker using candles to test for air leaks in an operating reactor started a fire that nearly caused a meltdown. The electrical signals to the pumps (and everything else) were lost. The practice of using candles to check for leaks in operating reactors has since been banned (gee... one would have thought...) but additional fire codes, created after the incident, have not been implemented at many reactors around the country -- including San Onofre!

Dick Cheney lied about the agenda and the participants of his secret his pro-nuclear energy policy (and many other things). Likewise, San Onofre's owner/operators lie about just about everything, too. Today (March 27th, 2012) a new report indicates that Southern California Edison misled the Nuclear Regulatory Commission -- and everyone else -- about the new steam generators. SCE said they were designed as exact replacements for the old steam generators. But it appears that in reality, their fluid flows were redesigned to increase output! But apparently instead, the new design increased their own wear and tear!

March 18, 2012
 
Dear Readers,
It's time to decommission San Onofre Nuclear Generating Station.  It's the only sensible thing to do.  It makes economic sense for just about everybody, and spares us the possibility of "Fukushima USA" here in SoCal.(Southern California) 
Right now, neither of San Onofre's two reactors are operating.  Southern California Edison is already predicting there could be rolling blackouts during the summer if they can't get the reactors running by then.  The threat of blackouts is at odds with the historic record of energy usage, which clearly shows that there is more than enough electrical generating capacity and transmission line capacity in SoCal to replace San Onofre. 
Nevertheless, SoCal residents can EXPECT rolling blackouts -- because SCE wants them to happen:  It may cost as much as a billion dollars (or more) to repair San Onofre.  Instead SCE could be securing contracts NOW for summer energy use.  They could be building a billion dollars' worth of solar rooftops, offshore wind turbines, turbine peaker plants, cogeneration plants, energy storage reservoirs, geothermal energy systems, etc..
But they don't want to, because when San Onofre is operating, it's "easy money":  A million dollars per day per reactor!  So instead they'll want to "prove" that SoCal "needs" San Onofre, so they won't prepare, the blackouts will happen, and then they'll expect us, the ratepayers, to pay for it all!
Meanwhile, there is still NO solution to the problem of long-term storage of nuclear waste, which has been piling up for decades in dangerous "temporary" locations at every nuclear power plant in the country (and nor will there ever be a good solution).  And NOR is there a solution to the dangers of nuclear weapons proliferation, which SanO exacerbates by producing plutonium and tritium.  Additionally, the dangers from terrorism, or from mother nature's fury, remain unsolved too.  San Onofre is built on or near several fault lines, and along the coast, nearly at sea level.  It's tsunami-prone and earthquake-prone.  And surrounded by about eight million people within 50 miles (noting that the U.S. Government recommended U.S. citizens evacuate from within 50 miles of Fukushima, Japan -- and ALSO noting that even that might not be far enough!). 
Furthermore, San Onofre continues to have problems with worker harassment (intimidation of workers to prevent them from reporting dangers) AND, paradoxically, worker safety complaints (that safe procedures aren't being followed).  San Onofre is officially (Nuclear Regulatory Commission's own data) the worst-run commercial nuclear facility in the nation on BOTH counts.
A meltdown at San Onofre would be the ruination of SoCal.  And we don't need a tsunami or "the big one" to cause it:  It's not inconceivable that a thing as simple as a flashlight dropped in the reactor water could start a cascade of failures, leading to a meltdown "just like" Chernobyl or Fukushima.  That's why they have regulations to prevent things like dropped flashlights (I mention this specifically because it happened there last month, and the contract worker, a temporary employee at the plant, who violated workplace rules by dropping the flashlight, then further violated the rules by trying to retrieve it -- and falling in!)
SCE doesn't want to be responsible for ANY of the costs to fix the reactors.  They just want to fix them any way they can, so they can restart them as soon as possible, so they can go back to making money -- and creating a ton a week of highly dangerous "spent fuel" which will be the real legacy of San Onofre's decades of operation: Millions of pounds of deadly poison sitting on our shoreline just waiting to be released by accident/sabotage, etc..  Southern California Edison will be long out of business, all of us will be long dead, California will be a nation unto itself (perhaps), but the waste will still be here.
The cause of San Onofre's current shutdown is defective replacement Steam Generators (SGs) made in Japan by Mitsubishi Heavy Industries.  MHI has been building SGs for nuclear power plants since the 1970s and have manufactured, shipped and installed well over 100 SG units around the world.  MHI's current annual report indicates they plan to double their nuclear steam generator business several times in the next three years to almost $10 billion annually.  So this is a big setback for them as well as for SanO's owners.  The problem is almost surely the result of incorrect manufacturing procedures: This didn't have to happen.  What other SGs around the world are in trouble?
Steam Generators are massive things used in Pressurized Water Reactors (PWRs).  PWRs have three coolant loops:  Water under very high pressure in the primary loop goes through the reactor core, gets heated (and irradiated), and then goes through thousands of very thin tubes inside the steam generators.
San Onofre's two SGs each have nearly 9,500 long, thin U-shaped tubes (the tube's walls are about the thickness of a credit card). Because the water inside the thin tubes is highly pressurized, it does not boil.  The water on the other side of the tubes (the secondary coolant loop) comes in contact with the hot tubes and turns to steam.  The steam is piped out of the containment domes and into the turbine room, where it is used to turn the turbines which generate electricity.  A third coolant loop (ocean water) condenses the steam in the second loop, and that condensate is then pumped back into the steam generators again.
When any company receives $800 million worth of equipment, it invariably inspects that new equipment very carefully to make sure it's exactly what they ordered.  San Onofre's replacement steam generators were inspected when they arrived at the plant in 2009, and found to be defective.  MHI had to send out a special team to SoCal to reweld them.  Like the defects that are appearing now, those defects SHOULD NEVER HAVE MADE IT OFF THE FACTORY FLOOR.
You can be sure numerous additional inspections were done after the problems were discovered in 2009. The additional inspections and repairs took about six months.  Then they put the steam generators in the reactors (two SGs in each of two reactors) and just over a year later... problems, problems, problems!
The first problems to show up were in Unit II, the older of the two operating reactors and the one to get its steam generators replaced first. Excessive wear was discovered on the thin U-shaped tubes inside the SGs, when were inspected during Unit II's first refueling outage after the SG replacement.  The outage was already far from "routine" despite repeated assurances by the utility that it was "just" a routine refueling outage:  The Reactor Pressure Vessel Head was being replaced, which is another massive (and expensive!) part which had worn out prematurely.  Neither the RPVH nor the SGs were ever supposed to wear out in the entire life of the plant. 
Unit II's SG wear is significant: Two tubes had at least 30% wear, and nearly 70 tubes had at least 20% wear, and about 700 tubes had at least 10% wear.  The new SGs are expected to last 40 years or longer -- but all of this excessive wear was detected after only about 14 months of operation!  PWRs rely on the SGs to remove excess heat from the reactor.  They are a vital safety component of the reactor, which is one reason there is a minimum of two SGs per reactor  (sometimes more than two) in every PWR in the world -- in case one fails.
San Onofre's engineers were quick to explain to the media and the public that the wear they found on Unit II was probably just wear from "settling in":  The parts merely had to "get comfortable" with each other.  I actually heard SanO employees using these "engineering" terms!
Then Unit III's steam generators failed, too -- and it was "discovered" the hard way:  A rupture  One of the nearly 20,000 tubes inside Unit III's SGs suddenly burst, and the subsequent release of primary coolant -- which is highly radioactive -- into the secondary coolant loop -- which normally isn't very radioactive -- caused the control room operators to have to shut down that reactor as well.  Some radiation was released to the atmosphere (and to the public) when the radioactive steam condensed back to water at atmospheric pressure.
Calling what happened Last January merely a "leak" is being too nice:  It was an extremely violent flashing to steam of super-heated, super-pressurized radioactive water and chemicals.  (If you passed your arm accidentally over the breach, it would take your arm off (by steaming it off!) in an instant.  (But at least the stump would be sanitized.))
Such steam generator tube ruptures are rare, and it's a good thing:  The real danger would be that one burst tube would damage the tube next to it, which would burst too, and so on in a cascade of failures that would throw metal parts throughout the primary and secondary coolant loops, damaging valves and reactor fuel assemblies, blocking water flow, and damaging the other SG.  And then what?  Fukushima USA: An inability to cool the reactor -- a meltdown.
San Onofre avoided that, but their troubles had only just begun.
After letting the reactor cool for several days, technicians went in and started trying to discover what had gone wrong with Unit III's new steam generators.  Was it "just" wear, like Unit II was experiencing?  It doesn't appear to be the same problem:  SanO employees identified 129 tubes that appeared to be excessively worn, and started pressure-testing them.  This involves increasing the pressure in one tube at a time to about three times the normal operating pressure.  Seven tubes have failed these tests already, and they've only just begun that phase of the testing!
Will it ever be safe, or reasonable, to restart these reactors with Mitsubishi Heavy Industry's steam generators?  When BOTH units are having problems?  (Exactly ONE tube in Unit II was pressure tested in light of the problems with Unit III.  That one tube passed the test.  All pressure tested tubes are plugged up permanently, and can no longer be used.)
There can be little doubt now that MHI has been delivering products with criminally-negligent workmanship, and San Onofre has been accepting those parts and using them.
One meltdown at SanO -- or two -- would destroy everything we love about SoCal.  Why spend billions of dollars just to restart THAT risk?  Right now we just have the spent fuel to deal with -- the radioactive waste pile.  It's deadly, difficult to manage, and will cost a fortune.  But at least it's NOT GROWING at the moment, and that's good.  In fact, slowly but surely, it's cooling and becoming less hazardous.
Restarting San Onofre is just plain stupid! 
Sincerely,
Ace Hoffman
Concerned Citizen
Carlsbad, CA
The author is an educational software developer.  His programs on mechanical pumps, the human heart, statistics and the periodic table are used in over 1000 universities around the world.