Oliver Smithies:[00:00:00] So here we are beginning Book δ, which starts on August 24th, 1982, and goes through October of the same year, beginning with further tests on materials isolated in the previous book.
A retest of the whole set, on Wednesday, August 25th, showing which were strongly positive and which were weakly positive of these. And screening, on page 9, Friday, August 27, continuing with a large number of positive colonies found by hybridization, [00:01:00] which then have to be characterized, as was going on Monday, August 30th, page 11, where probably 20 candidates were screened, and with the conclusion that — “Some very promising candidates. #84 is the best and #120 is good but weak,” etc. So continuing tests on the candidates, then. More screening — more screening, Wednesday, September 1st, Debbie Endean picking these, etc. Hybridizing and so forth. Very strongly positive, a whole bunch. [00:02:00] And then Cla digests of them, on the following page, and Sal digests, on page 17 and page 19, Saturday, September 4th and September 5th. Labor Day, September 6, Sal mix for nine samples, so on and so forth. Repeat, again, again, Sal digests, page 25, on Tuesday, September 7. “Conclusion: At last, 402 and 403 are OK.” [00:03:00] More minis, on page 27, with the conclusion “Abandon this. Wait for 84.”
And bulk preparation of a 5.6 kilobase Cla‑Cla fragment, which would include the suppressor gene and the beta globin sequence, next to [00:04:00] a little portion of the beta gene. A very faintly outlined map, showing the fragment that was being sought, suppressor gene next to a sequence of beta globin locus, ending partway into the beta globin gene, and then with a Sal‑Cla link. (laughs) Typical experiment, on 27, page 37, rather, Friday, September 17, with a comment “Very odd result” and “Abandon,” in large letters, “Too many weirdies.” But [00:05:00] on page 39, a more detailed map there shown, of various linkers that could be obtained from lambda, because of the need to get linkers. One couldn’t buy these, as I’ve said before. So a Sal-Cla-Sal linker, 499 base pair, a Cla-Sal-Cla linker, 973 bases, Cla-Sal-Exil, Cla‑‑ etc., etc., all these different linkers that we had to make, in order to assemble our construct. With more detail there. Which make — turn this ‑‑ and the maps would get more complicated but the [00:06:00] construct can eventually be made. Quite a lot of hard work, that would be very easy now. (long pause)
Preparative gels for some of the fragments continuing, page 43, Monday, September 20th. Continuing the same general tasks. [00:07:00] (pause) All these different little schemes to get various linkers. Thursday, September 23rd, 57, to get a Cla-Sal-Cla linker. “Conclusion: A trace better but not good enough to go on.” [00:08:00] (pause) Minis, minis, minis, Thursday, 29th, page 73. New Cla‑Cla digests.
And there is Clone 3, δ‑75, preparation. This is preparing the Cla‑Cla piece from δ‑79, Clone 3, a very large piece of DNA. [00:09:00] With the conclusion, on page 74, that “The new Cla portion didn’t help to increase the sample amount but both #12 and #17 still look good on Cla.” I was mistaken about that. The length of the Cla piece is shorter than that. Because it’s commented that “The Cla piece looks good at about 5.1 KB.” It should be a bit bigger, 5.2, in fact.[00:10:00] R1 and Cla digests of Clone 3 again, repeating, Saturday, October 2nd, page 81, just doing the same experiments over until one gets the correct fragment. Conclusion, on Sunday, October 3rd, “Cla+/‑Sal –” so-called new Cla, etc. — and the conclusion “At last, stable 2. Grow up 12‑2 for a big grow. Check the orientation first.”
New minis. [00:11:00] The orientation of the plasmid being considered, on page 87. One quite complex plasmid being considered on page 87. (pause) With mini orientation being [00:12:00] approached, on page 91, Tuesday, October first on the opposite side. “Conclusion: 12 and 17 are opposite orientation and the same size. Scrap 57.” So… The comment then, in big letters, “So, success at last. Either orientation is OK for further work. Select on the basis of the linker.” Clone 17, I think, will occur again, unless I’m mistaken. And here on the next page it is, indeed, page 93, Wednesday, October 6, with a fairly complete map now of what these fragments, and then 17 [00:13:00] being shown, with a Neo gene in it, and Clone 12 and delta-beta sequences with a supF gene.
Some tests of supF, on page 97, Thursday, October 7th, to see whether this supF will, in fact, allow the growth of DP50 supF. So here are various cultures, [00:14:00] grown up or found, C600, which is wild type, C82, KH82, which is not suppressor-minor, DH50 supF, which has two suppressor genes in it, and so on. And testing the launch for the ability to grow. Making sure that the suppressor gene will work in the way expected. Suppressor and Neo gene test, on 12‑2, on page 99, Friday, October 8th. [00:15:00] More tests of supF, on the following pages. Worrying about the orientation of the Neo gene relative to the promoters, on page 105, Monday, October 11th, with a conclusion that “Cla 17 looks better.” (pause)
Clone 3 being [00;16:00] looked at, on Tuesday, October 12. And the results on Tuesday, October 12th, page 109, “Fairly consistent with Clone 3 being OK,” from the left-hand — ”Prepare a large piece for cloning and other tests.” Clone 3 preparative gel, the Cla piece. (pause)
Large preparation in this experiment, “50mg of δ‑75 Clone 3, cut with Cla1,” and then on to a gel. And the results being shown on page 110, with “Excellent yields, ≥50%, and good purity.” The different fragments taken from that, which are called [1‑S?], 2, 3, and 4‑F, 1‑S being slow and 4 being fast, of these fragments, 1, 2, 3, 4, different fragments, Cla1, from δ‑75 Clone 3. [00:18:00] (pause)
Page 115, tests of supF working. “supF is OK in Cla 17, probably OK in Cla 12. And checks with C1a will confirm or not.” And those tests are done on the following page. Page 117, Thursday, October 14th, tests on C1a. “At least 106 suppression with Charon 3A delta lac‑‑” etc., “So the clone contains suppressor and it is a fairly good suppressor,” [00:19:00] 3.
More D‑‑ more DNA from a new 17. And on page 121 and 120, a map of the region, again, of the cosmid HT3 and of the Cla‑Cla fragments, EcoRI fragment — EcoR‑‑ fragment 5.2‑kilobase, which is the end of the clone that’s going to be used in the recombination, contains part of the delta globin gene. Five-point-two-kilobase fragment being shown there. Was a little obscure earlier, as to what 5.2 was.[00:20:00] Next stage considered, on page 121. (pause) Some possible diversion on constructing a different Xba vector from Charon 30A, a different vector of lambda bacteriophage, but with the conclusion that… This says, “Stick with Charon 3A.” [00:21:00] And on page 129, Saturday, October 16th, the various Xba vectors are being looked at. And the simplest is an EcoRI digest, “and insert a linker,” etc., etc. The capacity of the phages for isolating the different fragments that one might obtain. (pause) [00:22:00] (pause) The problem is the capacity of these vectors and the large construct contemplated. One of the constructs was going to result in a clone of 41,000 base pairs, 41 kilobases, another 39 kilobases, etc. So, continuing. [00:23:00] Sunday, October 17th, 133 page, making probes and linkers. Check of the ligations, on Monday, October 18th, page 139. More ligations, Monday, October 18. A probe, on 143, Tuesday, October 19th, with “#2 is correct and clean,” as a BamRI — making a [00:24:00] 1.93 kilobase probe.
And, page 145, “Paula‑‑” this Paula Henthorn, probably — “Paula’s packaging results show that both Charon 3A Xba and Charon delta-delta Xba –” that is Charon 3A with deletion — “packaged well enough for lifting and hybridization.” Because we’re about to start on a large series of experiments of having to package DNA and screen for bacteriophages that contain the recombinant fragment.[00:25:00] Looking again at Clone 12 and Clone 17, on page 147, Wednesday, October 20th. That, 12 and 17, both looked good. “Partial Cla1 digests needed,” on Thursday, October twenty‑first ‑‑ But it looked to have been too complete a digest. So repeat of Cla partials, on Thursday, October 21st, page 151, as we’re coming to the end of this book. Saturday, October 23rd, making buffers [00:26:00] and growing colonies — which is the end of Book δ, Saturday, October 23rd. [00:26:14]