Oliver Smithies:[00:00:00] Well, this is book little d that begins on February 5th, 1973 and goes through April 13th. So it’s really only one just two or three months’ book. But it has quite a few results. Still trying to work with lambda dv. February 5th, page one. And looking at what happens after adding the various things to the broth after growing. With the bacteria containing the lambda dv plasmid. Adding lysozyme and RNase and pronase in different orders. [00:01:00] And trying to notice what happened. The lysozyme. Some cells survived. Slow clearing. And then adding the RNase the viscosity didn’t change. And then adding pronase. And again comment. Possibly became hazier after the RNase. Definitely became clearer after the pronase. And continuing to retitrate some material again on the following page. And titer was quite nice, 1.3 times 10 to the 11. Lambda dv colony. [00:02:00] Marker rescue assay for lambda dv as I said earlier. I think it had a suppressor gene in the lambda dv, let’s see, colony d3A is now optical density 1.3. And others are similar. Therefore refrigerated after transferring, etc., etc. Should be OK. All close to the same. And titrating. And the two strains of bacteria, 594 and C600. One with the suppressor and one without. And 594 no colonies until 10 to the 5, and then [00:03:00] let me see. C600 had colonies. Not very clear here. Some comments that it wasn’t working as expected because — the comment but I put in 4 times 10 to the 7 to 10 ml, so 2 times 10 to the 6 per ml. Therefore no absorption. Evidently the assay didn’t work, though I was trying. [00:04:00] On February 7th, Wednesday trying various extractions with [bridge 58?] on page nine. Toluene [a bio dream?]. Seems good solvent of solid. So tested extraction, [50-50 bridge?] and toluene.
Also methylene dichloride very soluble, very mild as a surfactant, better than toluene. And carbon tetrachloride also a good solvent, no surface activity. [00:05:00] Continuing on the next page. Dichloromethane. Very milky top phase, indications of clearing, etc., so not efficient extraction. Try toluene and carbon tetrachloride, etc., etc. Lambda dv again on the following page, February 8th. Test with [bridge 5F?]. Deoxycholate was added. Lysozyme and magnesium and so on.[00:06:00] Drop of amyl alcohol and wheat germ lipase [quit hazard bridge 58?]. Conclusion. Return to [bridge?] and ethanol precipitation. So lambda dv again. At last we got an electron micrograph. This is lambda DNA. Lambda dv DNA. Spreading [for OS?]. And there is a beautiful image of lambda dv with the note on the spreading. But one good lambda dv and [00:07:00] bacteriophage DNA and lambda dv. The very satisfying images. At least I got some images of the bacteria.
So these images are very satisfactory to see. At least I got to see the lambda dv that I’ve made. I don’t think we shall ever see any replication [ports?] but who knows. I don’t remember seeing them. So lambda dv assay again trying to get that to work on page 16, 17. And the results were with different bacteriophage preparations. And the conclusion being in this case that only clone F carries a lambda exclusion factor. And that means that that gives a [00:08:00] difference whether it’s grown on one bacteria or on another. Only clone F.
There are some problems with that, other worries that are being talked about. Trying the DNA again on page 19. Lambda dv again DNA. And respread [in shadow?] on page 20. And one can see the DNA, that there is a lambda [00:09:00] dv DNA there, but it’s still got quite a bit of protein associated with it. Ross thinks that everything was OK. The trouble he diagnosed, this must be Ross Inman. The diagnosis of the trouble is too low a pH and/or a lack of formaldehyde. It clearly has lambda dv there but it’s very dirty compared with the other one. It’s Ross Inman’s high pH buffer being tried on page 23. Fairly complicated phage plan now attached to page 24. Bacteriophage plan, etc., etc.[00:10:00] Grow clones and do this and that and the other. Protocol. Fairly complicated bacteriophage genetics here being discussed on page 25. Test for temperature-inducible by growing, etc. Test for noninducible by immunity resistance. For example 10 to the 9 lambda c per plate and spot growth equal to resistance, etc., etc. [00:11:00] Experience that was later valuable. Rough DNA test for D. I don’t know who D is. Nuclei. Trypsin. TPCK. Following up on this scheme to try to get things under control on Saturday, March 3rd. New M9-gal glyc plus biotin plates. [00:12:00] Thinking about [511A?] and [511B?] reversion rate to gal insensitivity that is measurable. I don’t know why I wanted to get the reversion rate at this point. Reversion of [511A?] and [511B?] to gal resistance on Sunday, March 4th, page 35.
Should be about 2 times 10 to the 8 organism per plate. But once again no colonies. [00:13:00] Like the biotin. No growth. Should have had biotin there. d31 DNA again. d31 DNA, what’s that one? d31 DNA is nuclei. Trying some different substrates for looking at the DNA and with the comment that the Formvar carbon difficult to see and [spa?] mica ditto. But easy to see [00:14:00] good and plenty. So looks like mica is worth it. So you have to pay for mica versus this cheaper thing. Mica is a lot better. Lambda dv again on page 43. Spread down a [single wet ramp?]. Reading Saturday. [00:15:00] From the d3941 experiment. Batch on Thursday, March 8th, batch 49 mica and carbon test.
I must have been doing the [00:16:00] coating myself on the — because it says that the 1:00 p.m. coated [vaca?] less than 0.3 times 10 to the -5. And it was 0.6 times 10 to the -4 when it began and went to 1 times 10 to the -4 but [stop?] went down to about 2 [millimeters?] so I’m with somebody else doing the [fair vacuum actually get a high vacuum?] to do those experiments.
So lambda [C149?] or [C60?]. [00:17:00] About 2 times 10 to the 9 per plate on Friday, March 9th. From [Sid?].
Excuse me, Ollie.
Lambda dv. Anyway, clone on March 10th, 59. Trying to get new attempt at lysogen. With conclusion scrap them. Conclusions. These gal resistances are as likely to be revertant as true, [00:18:00] therefore continue with d59 approach. Which we’ll get to in a couple pages. Sunday, March 11th, new and repeat of lambda dv clone tests. And with the result with a nice checkmark. This is completely correct. Lambda dv must be there. The results being the all negative lambda [029P3?] on one plate, four out of six positive lambda [I21?], etc., etc. (inaudible) but anyway evidently feeling better about the assay. [00:19:00] And all are ultraviolet-sensitive on the next page. Prototrophs for d49. Decided to isolate single colony. Tuesday. All four. Got four colonies. All four are gal-sensitive and all are UV-sensitive. Test for UV sensitivity. This is this [M9-glu B1 plus bio?] plate of unknown at this point importance. Lambda dv again. [00:20:00] On page 61. So here’s a spreading on page 63 on mica and carbon. It looks like C slash mica. Carbon replica of mica I think is probably what that means. It’s not as nice as the previous one. So carbon mica versus the earlier one, which was on mica itself I think.[00:21:00] [00:22:00] So this is again looking at page 63 and 62 of book little d. Where there’s one nice lambda dv, though not as pretty as the earlier one. [00:23:00] I think these experiments must have been — these different tests of different bacteria strains must have been to try to get lambda to lyse without difficulty, but I can’t really understand them at this point. For example d66 parenthesis R4A plus lambda macrocolony test. Massive growth in the mucoid form. [00:24:00] Resistant to lambda [C60?] and [I21?]. Not likely to be the required lambda lysogen. Discard. Rough test of gal-sensitive to resistant. And lambda resistance. I see what it is. It’s the gal receptor. I remember now. The galactose receptor is needed for lambda attachment. And if the galactose gene is missing [00:25:00] then resistance to lambda infection occurs. That’s on Monday, March 19th, page 75 saying that clones must be gal-resistant, lambda [C60?] immune and [4C?] sensitive. Not very necessary. Lysogenic for lambda. Etc., etc., etc., etc. This permits spontaneous induction of [I4?] to infect [phi 11?]. Not immune to [I4?] which then allows the [00:26:00] [187 red minus excise minus?] to excise and give clear plaques. Which is not, etc. Complicated, I must say. Probably a bit too strong bacterial genetics for my taste at this stage.
DNA lambda dv again on Monday, Tuesday, March 20th, and on the following page. Again images that look to be circular DNA there but too long. [00:27:00] Then as it says the comment here is that lambda phage diluted 1 to 10 shows plenty of DNA and there were about a third are circles. Some with supercoiling. Some empty heads and tails. Clear difference in size. Go on therefore to test other bigger plasmids. So I think I’m of the opinion there that these are circular molecules all right, but they’re quite a lot bigger than the lambda dv molecules. They’re quite nice spreads. [00:28:00] Going on with titrating and so on. Test for gal [E?] transduction. More complicated ideas on page 93. R-factor from [Bob Brown?]. [00:29:00] More bacterial genetics. Saturday, March 24th test to prove that d95 colonies are transductant (inaudible) recombination [00:30:00] introducing the lambda into the bacterial chromosome to get a lysogen. And why I wanted the lysogen I’m not clear. It is recording, isn’t it?
Yeah. [00:31:00] An attempt on page 105, see R34 parenthesis R12 again, to see if one could get plasmid. With a comment that though there is an image of a longer plasmid than lambda dv the comment is that they are very sparse. [00:32:00] This is a bigger plasmid than lambda dv. More complicated tests. [00:33:00] Repeat of R12 DNA [rescue?] d107 R12 slash 2B culture from the [cold?] cooled in centrifuge, etc. (inaudible) lysozyme and then RNase and pronase. And then spread. There is a very long plasmid in there. [00:34:00] And some smaller one. Some quite small. Quite a few different sizes of plasmid. [00:35:00] More and more tests of this bacterial colony. [00:36:00] More and more tests. [00:37:00] Further plans discussed on page 145. Use P2 lytic plaques to isolate defective phages that are likely to be gal transducers. Pick lytic plaques from [W50S?] onto [X7734?] and assay for gal plus in order to see lytic centers. Use an extra heavy lawn of [X7734?] or use the new lytic lysate, etc. So difficulty. Coming to the end of this book and to the end of my understanding of the work. [00:38:00] April 6th, 73 and April 7th, etc. With the next to the last page saying inspection of d151, d149, etc., etc. All the questionable positives. In other words nothing convincing. Last attempt. Repick these. Also pick them on Sunday. On the cover pages of the book are some results. But no interpretation. [00:39:00] And so ends book d.