/
protocol.html
727 lines (592 loc) · 49.9 KB
/
protocol.html
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
<!DOCTYPE html>
<html lang="en" dir="ltr" class="client-nojs">
<head>
<meta charset="UTF-8" />
<title>Protocol</title>
<link rel="stylesheet" type="text/css" href="css/nav.css">
<link rel="stylesheet" type="text/css" href="css/painter.css">
<link rel="stylesheet" type="text/css" href="./css/new1.css">
<link rel="stylesheet" type="text/css" href="./css/page/protocol.css">
<script src="https://ajax.aspnetcdn.com/ajax/jquery/jquery-3.5.1.min.js"></script>
<script>
window.addEventListener('scroll', function() {
let top = window.scrollY
console.log(top)
if (top > 200) {
$('.main').css('transform', 'translateY(-300px)')
} else {
$('.main').css('transform', 'translateY(0%)')
}
})
</script>
</head>
<body>
<div id="loading">
<div class='loadingX' style="background-color: #abc2ea;">
<img src="./image/lodding_3.gif" height=100% width=100%>
</div>
</div>
<script>
document.onreadystatechange = function() {
if (document.readyState === "complete") {
setTimeout(
function() {
// document.querySelector("#loading").style.opacity = "0%";
document.querySelector("#loading").style.display = "none";
document.querySelector("#globalWrapper").style.display = "block";
}, 3000)
}
}
</script>
<div id='globalWrapper'>
<div id="content" class="mw-body" role="main" style="background-color: white">
<div id="HQ_page">
<div id="bodyContent">
<div id="mw-content-text" lang="en" dir="ltr" class="mw-content-ltr">
<section class="menu-section-area">
<!-- Navigation -->
<nav class="menu">
<div style="height: 0px;width: 0px;position: relative;">
<img src="./image/logo.png" width="50px" height="50px" style="position:absolute;top:-14px;">
</div>
<ol>
<li class="menu-item"><a href="./index.html">Home</a></li>
<li class="menu-item">
<a href="#0">Project
<svg version="1.1" class="plus-icon" xmlns="http://www.w3.org/2000/svg"
xmlns:xlink="http://www.w3.org/1999/xlink" viewBox="0 0 18 18">
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="10" y1="9" x2="17" y2="9" />
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="9" y1="9" x2="9" y2="1" />
<g id="lineGroup_1">
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="1" y1="9" x2="8" y2="9" />
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="9" y1="17" x2="9" y2="9" />
</g>
</svg>
</a>
<ol class="sub-menu">
<li class="sub-menu-item"><a href="./description.html">Description</a></li>
<li class="sub-menu-item"><a href="./design.html">Design</a></li>
<li class="sub-menu-item"><a href="./results.html">Results</a></li>
<li class="sub-menu-item"><a href="./supplement.html">Supplement</a></li>
<li class="sub-menu-item"><a href="./protocol.html">Protocol</a></li>
</ol>
</li>
<li class="menu-item"><a href="./lab_notebook.html">Lab notebook</a></li>
<li class="menu-item">
<a href="#0">Team
<svg version="1.1" class="plus-icon" xmlns="http://www.w3.org/2000/svg"
xmlns:xlink="http://www.w3.org/1999/xlink" viewBox="0 0 18 18">
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="10" y1="9" x2="17" y2="9" />
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="9" y1="9" x2="9" y2="1" />
<g id="lineGroup_1">
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="1" y1="9" x2="8" y2="9" />
<line fill="none" stroke-width="4" stroke-linecap="round"
stroke-miterlimit="10" x1="9" y1="17" x2="9" y2="9" />
</g>
</svg>
</a>
<ol class="sub-menu">
<li class="sub-menu-item"><a href="./team.html">Team</a></li>
<li class="sub-menu-item"><a href="./collaboration.html">Collaboration</a>
</li>
</ol>
</li>
</nav>
<!-- Navigation -->
</section>
<div class="img1"></div>
<div class="lnav">
<ul class="navul" style="margin-left: 0;padding: 0;">
<li id="nav1" class="uli cntli">
<a href="#h1">Materials</a>
</li>
<li id="nav2" class="uli cntli">
<a href="#h2">Methods</a>
</li>
<li id="nav3" class="uli cntli" style="height: 81px;">
<a href="#h3">Methods of PACE</a>
</li>
</ul>
</div>
<div class="content">
<div class="main" style="color: black;word-wrap: break-word;word-break: normal;">
<aside>
<div class="p1" style="word-wrap:break-word;word-break: normal;">
</br>
</div>
<div class="p2" id="h1">
</br>
<h1 style="text-align:left;line-height: 30px; ">
Materials</h1>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Biological materials
</h2>
<p style="text-indent: 0em;">
<i>E. coli</i> S2060 (Addgene, cat. no. 105064)
<br /><i>E. coli</i> S2208, pJC175e was transformed into S2060 to obtain the strain.
<br /><i>E. coli</i> C321 ΔA muts-T7
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;text-align:left;">
Plasmids
</h2>
<p style="text-indent: 0em;">
sfGFP Y66-S205C
<br />p15A-PBAD-Gen2-tRNACUA opt
<br />Mutagenesis plasmid MP6 (Addgene, cat. no. 69669)
<br />pJC175e (Addgene, cat. no. 79219)
<br />Accessory plasmid, including a tRNA sequence that recognises the TAG codon and the
<I>gⅢ</I> and LuxAB genes controlled by the T7 promoter.
<br />Complementary plasmid, including the T7 RNAP which contains two amber termination mutations.
<br />Selection plasmid, derived from bacteriophage M13 (V00604.2, ATCC 15669-B1) with the following modifications:
<br />Removal of <i>gⅢ</i>
<br /> Substitution of <i>gⅢ</i> RBS with synthetic RBS
<br /> Insertion of gene of interest immediately after synthetic RBS:AAAGAGGAGAAA
<br /> Insertion of the following sequence, containing an artificial promoter and RBS for production of downstream
<I>gVI</I>:
<br />GGCTCTAGAAGGAGATTTTCAACATGCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATCTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAATC
</p>
<div class="notebook_cell" style="text-align:center;">
<h2 style="font-size: 20px; margin-top: 30px;color: black;text-align:left;">
Primers</h2>
</br>
<table style="border:black 2px solid;text-align: center;align-items: center;margin: 0 auto;" border="1">
<tr>
<th>Name</th>
<th>Sequence 5'-3'</th>
</tr>
<tr>
<td>Gen 2-F</td>
<td>GCTAACAGGAGGAATTAACCATGGACGAATTTGAAATGATAAAGAG</td>
</tr>
<tr>
<td>Gen 2-R</td>
<td>CGGCATAGCGAAACTTAAAATTATAATCTCTTTCTAATTGGCTCTAAAATC</td>
</tr>
<tr>
<td>Vector-F</td>
<td>TTTTAAGTTTCGCTATGCCGG</td>
</tr>
<tr>
<td>Vector-R</td>
<td>GGTTAATTCCTCCTGTTAGCCCA</td>
</tr>
<tr>
<td>Reverse PCR-RBS-F</td>
<td>CACCTGCAGGTCTTTCTCCCTATAGTGAGTCGTATTAGTCGGCC</td>
</tr>
<tr>
<td>Reverse PCR-PROK-R</td>
<td>AGCAGGCTTTTTTGCATGTCATGCCAGTTCTAGCATAACCCC</td>
</tr>
<tr>
<td>G3-RBS-F</td>
<td>GAGAAAGACCTGCAGGTGCAGTAAGGAG</td>
</tr>
<tr>
<td>G3-PROK-R</td>
<td>AGGCATTTGAGAAGCACATTAGGTATATTCCGTGTGGTACTT</td>
</tr>
<tr>
<td>PROK-F</td>
<td>TGTGCTTCTCAAATGCCTGAG</td>
</tr>
<tr>
<td>PROK-R</td>
<td>CATGCAAAAAAGCCTGCTCGTTG</td>
</tr>
<tr>
<td>T7-P-F</td>
<td>CTACATAAATCAAACATAAGGAGGTAACCATGAACACGATTAACATCGCTAAGAAC</td>
</tr>
<tr>
<td>T7-P-R</td>
<td>GCTCTTCAGCCTTACGCGAACGCGAAGTCCG</td>
</tr>
<tr>
<td>AP-F</td>
<td>TCGCGTAAGGCTGAAGAGCTCATGCCAGTTCTAGCATAACCC</td>
</tr>
<tr>
<td>AP-R</td>
<td>TTATGTTTGATTTATGTAGTAGACTAGAAGAGCGTCGGCCTTACTTGCTAGCAG</td>
</tr>
<tr>
<td>AP-T7-F</td>
<td>AAGCAGAAGGCCATCCTGAC</td>
</tr>
<tr>
<td>AP-T7-R</td>
<td>GCGACACGGAAATGTTGAATAC</td>
</tr>
<tr>
<td>GOI-F</td>
<td>TAATGGAAACTTCCTCATGAAAAAGTCTTTAG</td>
</tr>
<tr>
<td>GOI-R</td>
<td>ACAGAGAGAATAACATAAAAACAGGGAAGC</td>
</tr>
<tr>
<td>pJC175e-Check-F</td>
<td>GCGGTATCATCAACAGGCTTA </td>
</td>
</tr>
<tr>
<td>pJC175e-Check-R</td>
<td>CGTTCCAGTAAGCGTCATACAT</td>
</tr>
<tr>
<td>CP-12S-F</td>
<td>CGACTTCTAGGACATCGAACTGGCTGCTATCC</td>
</tr>
<tr>
<td>CP-12S-R</td>
<td>CGATGTCCTAGAAGTCGTTCTTAGCGATGTTAATCG</td>
</tr>
<tr>
<td>CP-203-F</td>
<td>GTGGTCTTAGTGGCATAAGGAAGACTCTATTCATG</td>
</tr>
<tr>
<td>CP-203-R</td>
<td>TATGCCACTAAGACCACGCCTCGCCACC</td>
</tr>
</table>
</div>
</br>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Reagents </h2>
<p style="text-indent: 0em;">
2× YT Medium (Solarbio, cat. no. LA2470)
</br>YEAST EXTRACT (OXOID, cat. no. LP0021) TRYPTONE (OXOID,cat. no. LP0042) NaCl (HUSHI,cat. no. 10019318)
</br> SOC media (Solarbio, cat. no. LA2500)
</br>X-Gal (Solarbio, cat. no. R0404)
</br>Agar (Solarbio, cat. no. A8190)
</br>Ampicillin (Solarbio, cat. no. A7094)
</br>Chloramphenicol (Solarbio, cat. no. C8050)
</br> Tetracycline (Solarbio, cat. no. IT0130)
</br>Streptomycin (Solarbio, cat. no. S8290)
</br>L-arabinose (Solarbio, cat. no. A8060)
</br>Kanamycin (Solarbio, cat. no. K8020)
</br>lucose (Solarbio, cat. no. G8150)
</br> Anhydrotetracycline (Solarbio, cat. no. IA5330)
</br>IPTG (Isopropyl β-D- Thiogalactopyranoside ) (Solarbio, cat. no. I8070)
</br>Ascorbic acid (Solarbio, cat. no. A8100
</br>LBL medium
</br>TRYPTONE 10 g/L, YEAST EXTRACT 5 g/L, NaCl 5 g/L, 0.5 mg/ml ascorbic acid, pH=7.45, 33 ug/ml Chloramphenicol.
</br>DAM medium
</br>5g/L TRYPTONE, 2.5g/L yeast extract, 5g/L NaCl, 0.5mg/ml ascorbic acid, 10mM/L L-DOPA, 25uM/L Kanamycin, 2.96g/L IPTG.
</br>SapⅠ (New England BioLabs, cat. no. R0569S)
</br>T4 DNA ligase (New England BioLabs, cat. no. M0202V).
</br>FastPure Gel DNA (Nanjing Vazyme, cat. DC301-01)
</br>FastPure Plasmid Mini kit (Nanjing Vazyme, cat. DC201-01)
</br>DH5α Competent cell (Nanjing Vazyme, cat. C502-02)
</br>ClonExpress MultiS One Step Cloning Kit (Nanjing Vazyme, cat. C113-01)
</br>ClonExpress II One Step Cloning Kit (Nanjing Vazyme, cat. C112-01)
</br> 2×YT liquid media
</br> Add 2×YT media powder to a final concentration of 31 g/L in water. Mix to dissolve and then autoclave to sterilize at 121.0 °C. Store at room temperature indefinitely.
</br> 2×YT agar
</br>Add 2×YT media powder and agar to a final concentration of 31 g/L and 1.5% (wtol), respectively, in water. Autoclave to sterilize at 121.0 °C. Store at room temperature in the dark for up to 6 months.
</br>2×YT top agar
</br>Melt 2×YT agar using a microwave until completely liquid. Dilute hot 2×YT agar with room temperature 2×YT liquid media to a final agar concentration of 0.5% or 0.6%(wtol) and mix thoroughly.
</br>LB liquid media
</br>Add YEAST EXTRACT, TRYPTONE, NaCl to a final concentration of 5 g/L,10 g/L,10 g/L in water, respectively.
</br>LB agar
</br>Add YEAST EXTRACT, TRYPTONE, NaCl, agar to a final concentration of 5 g/L, 10g/L, 10g/L, 1.5% (wtol), respectively, in water.
</br> 2×TSS
</br>Add MgCl2, PEG 3350 and DMSO to LB media at final concentrations of 20 mM, 10% wtol and 5% volol, respectively, and sterilize using a Corning bottle-top vacuum filter system. Store at 4 °C indefinitely.
</br>5× KCM solution
</br>Add KCl, CaCl2 and MgCl2 to water at final concentrations of 100 mM, 30 mM and 50 mM, respectively. Store at room temperature indefinitely.
</br> DRM
</br>To 900 ml of water, add Harvard Custom Media A (18.1 g) and TWEEN-20 or TWEEN-80 (1 mL) and then autoclave to sterilize at 121.0℃. Allow to cool completely. Separately, add Harvard Custom Media C (5.9 g),
5 µl of 0.1 M CaCl2 solution and 6 µl of Trace Metals solution to 100 mL of water and sterilize using a Corning bottle-top vacuum filter system. Add the Harvard Custom Media C solution to the cooled Harvard
Custom Media A solution to make complete DRM. Store in the dark at 4 °C for up to 1 year.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Equipment
</h2>
<p style="text-indent: 0em;">
Biological shaker (Shanghai Zhichu Instruments, cat. no. ZHTY-50ES)
<br />Benchtop centrifuge (Thermo Fisher Scientific, cat. no. 75007201)
<br />Nano-100 microspectrophotometer(Hangzhou Allsheng Instruments, cat. no. AS-11010-00)
<br />UV-Vis spectrophotometer(Thermo Fisher Scientific, cat. no. Themo Genesys10s)
<br /> Electrophoresis(Beijing Liuyi, cat. no. DYY-6C)
<br />Absorbance Microplate Reader(TENCAN, cat. no.Tecan Infinite M200 NanoQuant)
<br />Isolated thermostatic incubator (Shanghai SENXIN , cat. no. GNP-9080)
<br />Gene Explorer(Bioer technology, cat. TC-96/G/H(b)B)
<br />13 mm, 0.22-μm PVDF syringe filter(0.22 µm; Merck Millipore Ltd., cat. no. PR05538)
<br />Gel imaging system(CLiN Qingxiang, cat. no. GenoSens2100)
</p>
</div>
<div class="p3 h2" id="h2">
</br>
<h1 style="text-align:left;line-height: 30px; ">
Methods</h1>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Preparation
</h2>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Preparation of competent cells</h3>
<p style="text-indent:0em;">
Frozen strains at -70℃ were inoculated into LB liquid medium(For S2060 and S2208 strains, use 2×YT liquid medium) by streaking method, labeled, and cultured overnight at 37℃. On the second day, a single colony was picked from the plate and inoculated
into a test tube containing 5ml LB liquid medium(For S2060 and S2208 strains, use 2×YT liquid medium) and incubated at 37℃ overnight by shaking; on the next day, 1mL of bacterial solution was inoculated into
a 500mL flask containing 100mL LB liquid medium(For S2060 and S2208 strains, use 2×YT liquid medium ) and incubated at 37℃ for about 4-6 hours (200-300 /min) by shaking violently. When the colony OD600 reaches
0.4-0.6, the bacterial solution is poured into a 50mL centrifuge tube under aseptic condition and centrifuged at 4000g for 10 minutes at 4℃. Discard the supernatant and add 2mL of pre-cooled LB liquid medium
to the centrifuge tube, mix gently, and suspend the bacteria. Add 2mL of pre-cooled 2xTSS to the centrifuge tube, mix thoroughly, divide it into 100μL parts, put them into 1.5mL centrifuge tube, and freeze them
with dry ice or liquid nitrogen. Store at -80 ℃.
</p>
</br>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Mention of plasmid
</h3>
<p style="text-indent:0em;">
According to the kit instructions for Nanjing Vazyme. </p>
</br>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">PCR fragment and vector
</h3>
<p style="text-indent:0em;">
The obtained plasmids are subjected to PCR using different programs and primers.
<br /> PCR Gen 2 fragment with Gen 2-F, Gen 2-R<br /> PCR pBAD33 vector with Vector-F, Vector-R<br /> After agarose gel electrophoresis, we extract DNA from the gel. The products are stored at -20 ℃ for subsequent
use.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Setting mutation conditions
</h2>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Error-prone PCR</h3>
<p style="text-indent:2em;">
Use StarMut Random Mutagenesis Kit. Multiple small system (20μL) amplification pre-experiments were carried out, setting a series of annealing temperature gradients and StarMut Enhancer gradients. According to the PCR results and the mutation rate in
the instructions. Final temperature (51.3℃) and Enhancer volume (6μL) were determined. </p>
<h3>Site-directed mutagenesis</h3>
<p style="text-indent:2em;">
With the help of two bioinformatic tools: fpocket and CAVER, we spot four mutational residues: Y32L, A67S, H70N, A167Q.Three pairs of degenerate primer (IUBcode)were designed to randomly introduce mutations in Gen 2, in which A67&H70 were too close to
be combined to one fragment.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Mutation
</h2>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Error-prone PCR</h3>
<p style="text-indent:2em;">
Using the error-prone PCR kit purchased from Gene-star company with (template plasmid 1, 2×StarMut Random PCR Mix 10μL, Gen 2-F, Gen 2-R 1μL each, Star Mut enhancer 6μL, sterile water 1μL, 53.1℃) conditions for error-prone PCR on MjTYR-Gen2 fragments.
The PCR products were verified by electrophoresis and purified using a kit purchased from Vazyme. The linearized vector and error-prone PCR fragments were then subjected to homologous recombination using homologous
recombination reagents purchased from Vazyme. </p>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Site-directed mutagenesis
</h3>
<p style="text-indent:2em;">
The whole pBAD33-Gen 2-Angew tRNA plasmid was divided into three parts and then amplified by three pairs of degenerate primers and 2 × Phanta Max Master Mix (Dye Plus) (Vazyme Biotech Co.,Ltd). The consequent three fragments with randomly introduced mutations
were then combined with ClonExpress MultiS One Step Cloning Kit (Vazyme), which is a cloning kit based on homologous recombination. E.coli C321 with pET28a-GfpS205C were used for subsequent chemical transformation.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Transformation
</h2>
<p style="text-indent:2em;">
Competent cells were thawed on ice. Pre-cooled plasmid mixture was added (each plasmid should
< 100 ng; Each transformation of up to 3 plasmids), fully mixed after 30min on ice, 42℃ water bath, heat shock 90s(For S2060 and S2208 strains, heat shock 75s). Put back on ice and stand for 2-3min; Add 500 microliters of LB liquid medium(For S2060 and
S2208 strains, use SOC medium), shake at 37℃, 200-300rpm/min, 1h; The bacterial solution was centrifuged at 8000rpm for 1min, the appropriate supernatant was removed, and then resuspended and coated in LB solid
medium(For S2060 and S2208 strains, use 2×YT solid medium) containing appropriate antibiotics and 1.5% agar at 37℃ for 16-18h. </p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Screening
</h2>
<p style="text-indent:2em;">
Transformants were selected from the mutational library plates, and each transformant was added with 1ml LBL medium (peptone 10g/L, yeast extract 5 g/L, sodium chloride 5g/L, 0.5 mg/ml ascorbic acid, pH=7.45, 100 μg/ mL amycin, 33 μg/ml chloramphenicol)
per well. 96-deep well plate was centrifuged at 300 rpm for 12h to prepare saturated bacterial solution. The saturated bacterial solution was then diluted 1:50 into a new 96-deep well plate containing
1ml antibiotic-free LBL medium per well and incubated at 300rmp for 2h in shock. At this time, the OD value of bacterial solution was close to 0.4, 100 μL of DOPA solution (10 mM/L L-DOPA, 0.5mg/ml ascorbic
acid) was added to each well, and then the culture was incubated for 2 hours with 300 rmp shock. Then 100ul DAM (5 g/L peptone, 2.5 g/L yeast extract, 5 g/L sodium chloride, 0.5 mg/ml ascorbic acid,
10ml L-DOPA, 25uM/L kanamycin, 0.23 μM/L, 2.96 g/L IPTG) was added to each well. The incubation was continued at 300rmp for 4h to obtain the induced bacterial solution.
</p>
<p style="text-indent:2em;">The induced bacterial solution was centrifuged at 3000 rmp for 30 min, and the supernatant was discarded. Each well was blown and resuspended with 200ul cold PBS buffer, and 100 μL was taken into a 96-well
fluorescence plate and measured with a microplate reader. Measurement indicators include Ex=450nm/Em=500nm fluorescence, Ex=535nm/Em=585nm fluorescence and OD 600 light absorption value.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Characterization
</h2>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Analog analysis
</h3>
<p style="text-indent:2em;">
For the mutant, we modeled it by AlphaFold2. The simulation process was completed on the cloud server (Shenzhen Bkunyun Cloudcomputing Co.,Ltd.). After the simulation, we selected the model with the highest score as the protein model for our next molecular
dynamics simulation. The molecular dynamics simulations were done using Gromacs, Among them, Energy minimization, Ensemble Equilibration and final molecular dynamics simulation was completed on the cloud
server (Shenzhen Bkunyun Cloudcomputing Co.,Ltd.), and the remaining steps were performed locally. In the simulation, OPLS-AA/L All-Atom Force Field (2001 Aminoacid Dihedrals) was selected as the force
field, and SPC/E Water Force field was selected as the water molecular force field.
</p>
<h3 style="font-size: 17px; margin-top: 30px;color: black;">Validation test
</h3>
<p style="text-indent:2em;">
The mutants with high orange fluorescence value were selected and the plasmids were transferred into the competent state again, and the fluorescence value was measured whether it was consistent with that before.
</p>
</div>
<div class="p4 h3" id="h3">
</br>
<h1 style="text-align:left;line-height: 30px; ">
Methods of PACE </h1>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Golden Gate cloning
</h2>
<p style="text-indent:2em;">
SP was cloned by Golden Gate assembly.</p>
<p>1. For Golden Gate assembly,SapI (New England BioLabs), was used as the type IIS restriction enzymes along with T4 DNA ligase (New England BioLabs). </p>
<p>2. Typical assemblies contained final concentrations of ~0.5–2 ng kb−1 µl−1 plasmids, with a ~2/1 ratio of donor to acceptor plasmids (splitC and splitD).
</p>
<p>3. Assemblies were thermally cycled (5 min 37℃ , 5 min 16℃ for 30 cycles, 5 min 60°C ,4℃) and followed by transformation into chemically competent cells (Steps 4-7).
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Phage cloning
</h2>
<p style="text-indent:2em;">
4. Transform phage genomes assembled through Golden Gate cloning method into S2208 cells and substituting the plasmid used in Step 1 with the entire yield of assembled phage genome.
</p>
<p>5. After the heat shock step, put the cell mixture back on ice and stand for 2-3 min.
</p>
<p>6. Dilute the cell mixture into 10 ml of antibiotic-free 2× YT liquid media in a culture tube.
</p>
<p>7. Pellet 2 ml of the resultant culture by centrifugation at 8000 g for 3 min.
</p>
<p>8. Collect and filter the supernatant using a 3-ml syringe fitted with a 13-mm, 0.22-μm PVDF syringe filter to remove residual cells. Collected phage may be clonally isolated (Steps 23–25) using plaque assays (Steps
18–22) and sequenced (Steps 26–29).
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Activity-independent phage plaque assays
</h2>
<p style="text-indent:2em;">
9. Dilute a saturated culture of S2208 cells in 2×YT liquid media supplemented with ampicillin and grow to an OD600 of 0.6–0.9 at 37℃ in a biological shaker.</p>
<p>10. Dilute phage serially in water in three 100-fold increments to yield four total samples (undiluted and 102-, 104- and 106-fold diluted). </p>
<p>11. Transfer 10 μl of each dilution into microcentrifuge tubes. </p>
<p>1.2× YT top agar. To each phage dilution, add 150 μl of diluted S2208, add Bluo-Gal to 2× YT top agar at a final concentration of 0.04%, stand for 2-3 min, add 1 ml of warm (~55 ℃)2×YT top agar. </p>
<p>12. After mixing, plate each phage dilution onto one quadrant of a Petri X-plate containing 2 ml of solidified 2×YT agar in each quadrant. </p>
<p>13. Incubate plates overnight at 37 °C. </p>
<p>14. Calculate the phage titer by the following formula: titer (in pfu/ml) = (# of plaques in quadrant) × (dilution factor of quadrant) × 100.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Isolation of clonal phage
</h2>
<p style="text-indent:2em;">
15. Pick a single plaque from a plaque assay plate by touching a P10 pipette tip to the surface of the top agar.
</p>
<p>16. Place the pipette tip into 2-3 ml of DRM and grow at 37 °C in a biological shaker for 16-20 h.
</p>
<p>17. Pellet the cells by centrifuging at 8000 g for 2 min, collect and filter the supernatant using a 3-ml syringe fitted with a 13-mm, 0.22-μm PVDF syringe filter to remove residual cells.
</p>
<p>18. Determine the phage titer using an activity-independent phage plaque assay (Steps 9-15)
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
Sequencing clonal phage
</h2>
<p style="text-indent:2em;">
19. Pick a single plaque from a plaque assay plate by touching a P10 pipette tip to the surface of the top agar.
</p>
<p>20. Amplify the phage DNA by using PCR amplify the POI insert on the phage (Place the tip into a PCR reaction mixture. Then, let the pipette tip from Step 20 sit in the reaction for at least 30 s before removing.
After running the PCR reaction, submit PCR product directly for Sanger sequencing.).
</p>
<p>21. Place the pipette tip from (Step 16) in a culture tube containing 3 ml of DRM and incubate in a biological shaker at 37℃ for 16-20 h.
</p>
<p>22. Pellet the cells by centrifuging at 8,000g for 2 min and then collect and filter the supernatant using a 3-ml syringe fitted with a 13-mm, 0.22-μm PVDF syringe filter to remove residual cells.
</p>
<h2 style="font-size: 20px; margin-top: 30px;color: black;">
MP6 mutation rate assay
</h2>
<p style="text-indent:2em;">
23. MP6 was transformed into S2060 and plated on Chl-resistant 2×YT plates containing 100mM D-Glucose.
</p>
<p>24. Monoclones were picked and shaken till saturation in 2×YT medium containing 25 mM D-Glu and Chl;
</p>
<p>25. The above bacterial solution was diluted 103-fold and grown to OD600 of 0.5-0.7. Grown to mid-log phase.
</p>
<p>26. Samples were divided into two: one with 25 mM D-Glucose and the other with 25 mM arabinose, and incubated for 12h until saturation was induced.
</p>
<p>27. The saturated solution was diluted by gradient and applied to 2×YT plates containing 100 mM D-Glucose and Chl and the saturated solution was applied to 2×YT plates containing 100 mM D-Glucose and Rif 37°C for
18-24 h.
</p>
<p>28. The number of colonies of each culture on D-Glucose ± Rif plates was counted. Formula for calculating MP6 mutation rate (tbp: substituents per bp per generation): ubp = f/[R × ln(N//N0)].
</p>
<h2 style="font-size:20px; margin-top: 30px;color: black;">
Luciferase assay
</h2>
<p style="text-indent:2em;">
29. AP was transformed into S2060 competent cells(Steps 4-7). It coupled bacterial luciferase (LuxAB) with <i>gⅢ</i> translationally.
</p>
<p>30. Pick a single colony into DRM supplemented with Ampicillin and Streptomycin and incubate it in a biological shaker at 37℃ for 16-20 h.
</p>
<p>31. Make 100 times dilution of the saturated culture of host cells into DRM and incubate in a biological shaker at 37℃ until the OD
<SUB>600</sub> of diluted culture reaches 0.4-0.6.
</p>
<p>32. Infect the host cells with phage. Incubate cells in a biological shaker at 37℃ for 1-2 h.
</p>
<p>33. Read luminescence using a plate reader with the following settings:
</p>
<p>(1) Set temperature to 37℃.
</p>
<p>(2) Shake (orbital) for 10 s with 1-mm amplitude and 582 r.p.m.
</p>
<p>(3) Read absorbance at 600 nm (number flashes = 10, settle time = 0 ms).
</p>
<p> (4) Read luminescence (attenuation = none, settle time = 0 ms, integration time = 500 ms).
</p>
</div>
</aside>
</div>
</div>
</div>
</div>
</div>
</div>
<div class="footer " style="position:absolute;right:0;left:0;margin:0 auto;width: 100%;height: 400px;background-color: black;margin-top: -200px;position: relative;">
<div class="container" style="width:100%;height:100%;background-color: black;width: 100%;font-size: 14px;">
<div class="footer-img" style="position:absolute;left: 0;">
<img src="./image/footer.jpg" width="600px" height="230px">
</div>
<div class="footer-content" style="text-align: center;right: 0;height: 100%;width: 50%;background-color: black;top: 0;">
<ul class="footer-content-ul">
<li class="first">
<a href="./index.html" style="font-size: 21px;">HOME</a>
</li>
</ul>
<ul>
<li class="first">
<a href="./description.html" style="font-size: 21px;">PROJECT</a>
</li>
<li>
<a href="./description.html">Description</a>
</li>
<li>
<a href="./design.html">Design</a>
</li>
<li>
<a href="./results.html">Results</a>
</li>
<li>
<a href="./supplement.html">Supplement</a>
</li>
<li>
<a href="./protocol.html">Protocol</a>
</li>
</ul>
<ul class="footer-content-ul">
<li class="first">
<a href="./lab_notebook.html" style="font-size: 18px;">LAB NOTEBOOK</a>
</li>
</ul>
<ul class="footer-content-ul">
<li class="first">
<a href="./team.html" style="font-size: 21px;">TEAM</a>
</li>
<li>
<a href="./team.html">Team</a>
</li>
<li>
<a href="./collaboration.html">Collaboration</a>
</li>
</ul>
</div>
<div class="footer-row">
<div class="col-md-12 " style="margin-top: -30px; ">
<p style="text-align: center;font-size: 16px; color: white;">Copyright © 2022 NAU All rights reserved.
</p>
</div>
</div>
</div>
</div>
<script src="./js/longli.js"></script>
</html>