Also download the pBR322 gene (pbr322.seq) and cut out just the gene sequence (pbr322.sq1).
Do the same for the cloning vector pBR322.DOS>gene4 <firefly8.res >firefly8.out
DOS>gene4 <pbr322.res >pbr322.out
Luciferase pBR322
---------------------------
ag/ct Alu I
137 15
260 30
371 686
453 1089
609 1997
1022 2054
1879* 2065
2114
2133
2414
2640
2730
2776
3033
3554
3654
3717
a/cgt Mae II
646 901
942 957
1604 1546
1944* 1570
1970* 1800
2226
3176
3592
3965
4285
t/taa Mse I
412 34
574 56
786 1720
867 1940
1066 1972
1098 2254
1395 3179
1579 3231
1690 3236
1765* 3250
1771* 3303
1871* 3538
1883* 3577
1899* 3942
1985* 4314
t/cga Taq I, TthHB8 I
31* 24
976 339
985 652
1278 1127
1378 1268
1840* 2573
4017
c/t*ag Dde I*
739 1581
1786* 1743
2283
2748
3157
3323
3863
4289
g/a*tc Hinf I*
727 632
1437 852
1892* 1006
1304
1525
2029
2373
2448
2844
3361
/gt*ac Mae III*
16* 125
199 213
498 881
720 1148
1090 1808
1737* 1831
1935* 1915
2128
2223
2830
2893
3009
3292
3623
3681
3834
4022
tt/cgaa Asu II, BstB I
30* none
ttt/aaa Dra I
1764* 3230
1882* 3249
3941
g/aattc EcoR I
2* 0
142
662
---------------------------
Luciferase pBR322
---------------------------
31* 24
976 339
985 652
1278 1127
1378 1268
1840* 2573
4017
---------------------------
Since complete digestion splits the coding section of the luciferase gene, thus destroying it, this is obviously undesirable. On the other hand, partial digestion may give a chance of preserving the luciferase gene intact. The 7*8/2=28 possible segments from partial digestion are listed below. Note that for a linear DNA, the number of possible fragments from n restriction sites is n*(n+1)/2.-------------------------------- Segment Start End # bases ------------------------------- A 1 30 30 B 31 975 945 C 976 984 9 D 985 1277 293 E 1278 1377 100 F 1378 1839 462 G 1840 2009 170 ------------------------------- Total 2009 (check)
We can use a program to find a list of the sizes in a partial digest.
Of the 28 possibilities above, only one ("BCDEF") contains the complete luciferase gene (from base 69 to base 1715).-------------------------------- Segment Start End # bases -------------------------------- A 1 30 30 B 31 975 945 C 976 984 9 D 985 1277 293 E 1278 1377 100 F 1378 1839 462 G 1840 2009 170 AB 1 975 975 BC 31 984 954 CD 976 1277 302 DE 985 1377 393 EF 1278 1839 562 FG 1378 2009 632 ABC 1 984 984 BCD 31 1277 1247 CDE 976 1377 402 DEF 985 1839 855 EFG 1278 2009 732 ABCD 1 1277 1277 BCDE 31 1377 1347 CDEF 976 1839 864 DEFG 985 2009 1025 ABCDE 1 1377 1377 BCDEF* 31 1839 1809 CDEFG 976 2009 1034 ABCDEF 1 1839 1839 BCDEFG 31 2009 1979 ABCDEFG 1 2009 2009 --------------------------------
-------------------------------
Segment Start End # bases
-------------------------------
a 24 338 315
b 339 651 313
c 652 1126 475
d 1127 1267 141
e 1268 2572 1305
f 2573 4016 1444
g 4017 4361 368
1 23
-------------------------------
Total 4361 (check)
We can use a computer program to find a list of the sizes in a partial digest.
Since we want to preserve the entire machinery (ORI, promoter/operator, selection markers, etc.), usually only the plasmid DNA with a single cut is of main interest to us.-------------------------------- Segment Start End # bases -------------------------------- a 24 338 315 b 339 651 313 c 652 1126 475 d 1127 1267 141 e 1268 2572 1305 f 2573 4016 1444 g 4017 23 368 ab 24 651 628 bc 339 1126 788 cd 652 1267 616 de 1127 2572 1446 ef 1268 4016 2749 fg 2573 23 1812 ga 4017 338 683 abc 24 1126 1103 bcd 339 1267 929 cde 652 2572 1921 def 1127 4016 2890 efg 1268 23 3117 fga 2573 338 2127 gab 4017 651 996 abcd 24 1267 1244 bcde 339 2572 2234 cdef 652 4016 3365 defg 1127 23 3258 efga 1268 338 3432 fgab 2573 651 2440 gabc 4017 1126 1471 abcde 24 2572 2549 bcdef 339 4016 3678 cdefg 652 23 3733 defga 1127 338 3573 efgab 1268 651 3745 fgabc 2573 1126 2915 gabcd 4017 1267 1612 abcdef 24 4016 3993 bcdefg 339 23 4046 cdefga 652 338 4048 defgab 1127 651 3886 efgabc 1268 1126 4220 fgabcd 2573 1267 3056 gabcde 4017 2572 2917 abcdefg 24 23 4361 under control of P1 promoter bcdefga 339 338 4361 within tetr, under P2 cdefgab 652 651 4361 within tetr, under P2 defgabc 1127 1126 4361 within tetr, under P2 efgabcd 1268 1267 4361 within tetr, under P2 fgabcde 2573 2572 4361 maybe too far away from promoters gabcdef 4017 4016 4361 within beta-lactamase, under P1 & P3 --------------------------------
The restriction enzyme Taq I cuts pBR322 at 7 separate positions. As mentioned above, with a smaller number of cuts, we get a much smaller number of possible digest fragments, which, in turn, leads to a much smaller number of possible recombinations later during the ligation step and a much higher probability of isolating the desired recombination. Optimally, one single cut made at the right place (e.g., within an existing unneeded protein or immediately after a promoter in the cloning vector) is the best. If we are willing to purchase another restriction enzyme, then we might also want to consider the following ones that have recognition sites different from Taq I yet produce the same exposed cohesive ends (i.e., 5'-cg...-3'). Such an enzyme may find more suitable sites in pBR322. Such an enzyme can be identified by searching for "/cg" through a list of commercially available restriction enzymes with a text editor. The following lists the restriction enzymes that fit the description along with the recognition sites.
gt/cgac Acc I* (will also cut gt/atac at 2244) 651 at/cgat Cla I 23 ga/cgtc gg/cgcc BsaH I* 413 434 548 1205 4284 g/cgc HinP I 101 233 261 414 435 495 549 701 776 816 947 1206 1357 1419 1455 1645 1728 2074 2177 2207 2348 2381 2651 2718 2818 2992 3101 3494 3587 3924 4256 c/cgg Hpa II, Msp I 161 170 387 402 411 534 694 770 930 1020 1258 1284 1485 1665 1812 2119 2153 2680 2827 2853 3043 3447 3481 3548 3658 3900 gg/cgcc Nar I 413 434 548 1205Specifically, Acc I and Cla I make a single cut at base 651 (within tetr) and base 23 (under control of P1 promoter), respectively. These are good candidates if a different enzyme is employed to digest pBR322.
Fusion product of "BCDEF" (31-1839), the only firefly luciferase digest segment with an intact gene, with one of the following pBR322 fragments may give what we are after. These represent making a single cut on the circular plasmid and inserting the linear luciferase gene into that position.
-----------------------------------------------------------
Circular Bases from Bases from Insertion Point
Recombinant pBR322 Firefly &
Plasmid ----------- ---------- Controlling Promoter
Start Stop Start Stop
-----------------------------------------------------------
1 abcdefg-BCDEF- 24 23 31 1839 under control of P1 promoter
2 bcdefga-BCDEF- 339 338 31 1839 within tetr, under P2
3 cdefgab-BCDEF- 652 651 31 1839 within tetr, under P2
4 defgabc-BCDEF- 1127 1126 31 1839 within tetr, under P2
5 efgabcd-BCDEF- 1268 1267 31 1839 within tetr, under P2
6 gabcdef-BCDEF- 4017 4016 31 1839 within beta-lactamase, under P1 & P3
-----------------------------------------------------------
Case 1. The insertion point of the first case above (at base 24 of pBR322) is located before the P2 promoter and cannot be controlled by it. This insertion point may seem to be located upstream from the P1 promoter. However, since the P1 promoter is located on the other strand that reads in the reverse direction, this insertion point is actually downstream to the P1 promoter. Thus, this case should work, and the resulting plasmid should be resistant to both tetracycline and ampicillin. Furthermore, with this construct, we can produce beta-lactamase alone without luciferase by inducing P3, but we cannot produce luciferase alone without co-producing beta-lactamase because they are both controlled by the same P1 promoter.
Case 2. On the other hand, if the luciferase gene is inserted into the site of tetracycline resistance protein or that of beta-lactamase, we need to check the gene sequence to make sure that there is no frame-shift that destroys luciferase codons.
For example, inserting the luciferase gene into the first restriction site within tetr (at base 339 of pBR322) gives the following sequence (pbr-luc8.sq1), where the tetr and luciferase genes are marked by capital letters for easy identification).
pBR 1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
pBR 61 ttgctaacgc agtcaggcac cgtgtATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
pBR 121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
pBR 181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
pBR 241 TGCGTTGATG CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
pBR 301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTAT
fire 1 cgaagtccc taaacggtag aggaaaagtt
fire 61 tttgaaaaAT GGAAATGGAA AAGGAGGAGA ATGTTGTATA TGGCCCTCTG CCATTCTACC
fire 121 CCATTGAAGA AGGATCAGCT GGAATTCAGT TGCATAAGTA CATGCATCAA TATGCCAAAC
fire 181 TTGGAGCAAT TGCTTTTAGT AACGCCCTTA CTGGAGTTGA CATTTCTTAC CAAGAATACT
fire 241 TTGATATTAC ATGTCGTTTA GCTGAGGCCA TGAAAAACTT TGGTATGAAA CCGGAAGAAC
fire 301 ATATTGCTTT GTGCAGTGAA AATTGTGAAG AATTTTTCAT CCCTGTACTT GCTGGTCTTT
:
... continued ...
The first start codon "ATG" marked blue above comes from the
tetracycline resistance protein gene originating from pBR322.
The second start codon "ATG" comes from the firefly luciferase
gene. Note that "ACT" marked green above is the last complete
codon from the original pBR322. Two bases "AT" from pBR322 and a
third base "c" from firefly combine to form a new codon. Because
of the frame-shift (shift in 3-letter codons) introduced at the
restriction site, the tetracycline resistance protein originating
from pBR322 is terminated prematurely at the "tag" sequence
originating from the firefly. Fortunately, this allows a fresh
reading of luciferase's start codon "ATG" and an unaltered
expression of the luciferase sequence. Computer analysis of the
above sequence (pbr-luc8.sq1) will also
show the expression of a viable luciferase.
DOS>gene4 <pbr-luc8.res >pbr-luc8.outThe rest of the tetr gene (fragments b, c, and d) is not required; thus, the following combinations also give rise to viable luciferase. Conceptually, this corresponds to cutting out the tetr gene and inserting the luciferase gene in its place. The last one is the most compact, but there is also no harm in leaving useless pieces of tetr gene around.
-----------------------------------------------------------
Circular Bases from Bases from Insertion Point
Recombinant pBR322 Firefly &
Plasmid ----------- ---------- Controlling Promoter
Start Stop Start Stop
-----------------------------------------------------------
bcdefga-BCDEF- 339 338 31 1839 within tetr, under P2
cdefga-BCDEF- 652 338 31 1839 within tetr, under P2
defga-BCDEF- 1127 338 31 1839 within tetr, under P2
efga-BCDEF- 1268 338 31 1839 within tetr, under P2
-----------------------------------------------------------
Case 3. Now, let's examine inserting the luciferase gene at the second restriction site within tetr (at base 652 of pBR322). This recombination gives the following sequence (pbr-luc8.sq2).
pBR 1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
pBR 61 ttgctaacgc agtcaggcac cgtgtATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
pBR 121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
pBR 181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
pBR 241 TGCGTTGATG CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
pBR 301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA TCATGGCGAC
pBR 361 CACACCCGTC CTGTGGATCC TCTACGCCGG ACGCATCGTG GCCGGCATCA CCGGCGCCAC
pBR 421 AGGTGCGGTT GCTGGCGCCT ATATCGCCGA CATCACCGAT GGGGAAGATC GGGCTCGCCA
pBR 481 CTTCGGGCTC ATGAGCGCTT GTTTCGGCGT GGGTATGGTG GCAGGCCCCG TGGCCGGGGG
pBR 541 ACTGTTGGGC GCCATCTCCT TGCATGCACC ATTCCTTGCG GCGGCGGTGC TCAACGGCCT
pBR 601 CAACCTACTA CTGGGCTGCT TCCTAATGCA GGAGTCGCAT AAGGGAGAGC GT
fire 1 cgaagtccc taaacggtag aggaaaagtt
fire 61 tttgaaaaAT GGAAATGGAA AAGGAGGAGA ATGTTGTATA TGGCCCTCTG CCATTCTACC
fire 121 CCATTGAAGA AGGATCAGCT GGAATTCAGT TGCATAAGTA CATGCATCAA TATGCCAAAC
fire 181 TTGGAGCAAT TGCTTTTAGT AACGCCCTTA CTGGAGTTGA CATTTCTTAC CAAGAATACT
fire 241 TTGATATTAC ATGTCGTTTA GCTGAGGCCA TGAAAAACTT TGGTATGAAA CCGGAAGAAC
fire 301 ATATTGCTTT GTGCAGTGAA AATTGTGAAG AATTTTTCAT CCCTGTACTT GCTGGTCTTT
:
... continued ...
This time, the last codon "CGT" marked green above occurs right
at the boundary, with no residual bases hanging over from pBR322.
However, tetr is prematurely terminated because of the
"taa" stop codon from the firefly. This, too, leads to a fresh
reading of the luciferase gene and results in an unaltered
luciferase sequence.
Case 4. Now, let's examine inserting the luciferase gene at the third restriction site within tetr (at base 1127 of pBR322). This recombination gives the following sequence (pbr-luc8.sq3).
pBR 1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
pBR 61 ttgctaacgc agtcaggcac cgtgtATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
pBR 121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
pBR 181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
pBR 241 TGCGTTGATG CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
pBR 301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA TCATGGCGAC
pBR 361 CACACCCGTC CTGTGGATCC TCTACGCCGG ACGCATCGTG GCCGGCATCA CCGGCGCCAC
pBR 421 AGGTGCGGTT GCTGGCGCCT ATATCGCCGA CATCACCGAT GGGGAAGATC GGGCTCGCCA
pBR 481 CTTCGGGCTC ATGAGCGCTT GTTTCGGCGT GGGTATGGTG GCAGGCCCCG TGGCCGGGGG
pBR 541 ACTGTTGGGC GCCATCTCCT TGCATGCACC ATTCCTTGCG GCGGCGGTGC TCAACGGCCT
pBR 601 CAACCTACTA CTGGGCTGCT TCCTAATGCA GGAGTCGCAT AAGGGAGAGC GTCGACCGAT
pBR 661 GCCCTTGAGA GCCTTCAACC CAGTCAGCTC CTTCCGGTGG GCGCGGGGCA TGACTATCGT
pBR 721 CGCCGCACTT ATGACTGTCT TCTTTATCAT GCAACTCGTA GGACAGGTGC CGGCAGCGCT
pBR 781 CTGGGTCATT TTCGGCGAGG ACCGCTTTCG CTGGAGCGCG ACGATGATCG GCCTGTCGCT
pBR 841 TGCGGTATTC GGAATCTTGC ACGCCCTCGC TCAAGCCTTC GTCACTGGTC CCGCCACCAA
pBR 901 ACGTTTCGGC GAGAAGCAGG CCATTATCGC CGGCATGGCG GCCGACGCGC TGGGCTACGT
pBR 961 CTTGCTGGCG TTCGCGACGC GAGGCTGGAT GGCCTTCCCC ATTATGATTC TTCTCGCTTC
pBR 1021 CGGCGGCATC GGGATGCCCG CGTTGCAGGC CATGCTGTCC AGGCAGGTAG ATGACGACCA
pBR 1081 TCAGGGACAG CTTCAAGGAT CGCTCGCGGC TCTTACCAGC CTAACTT
fire 1 cgaagtccc taaacggtag aggaaaagtt
fire 61 tttgaaaaAT GGAAATGGAA AAGGAGGAGA ATGTTGTATA TGGCCCTCTG CCATTCTACC
fire 121 CCATTGAAGA AGGATCAGCT GGAATTCAGT TGCATAAGTA CATGCATCAA TATGCCAAAC
fire 181 TTGGAGCAAT TGCTTTTAGT AACGCCCTTA CTGGAGTTGA CATTTCTTAC CAAGAATACT
fire 241 TTGATATTAC ATGTCGTTTA GCTGAGGCCA TGAAAAACTT TGGTATGAAA CCGGAAGAAC
fire 301 ATATTGCTTT GTGCAGTGAA AATTGTGAAG AATTTTTCAT CCCTGTACTT GCTGGTCTTT
:
... continued ...
This time, there is one residual base hanging on pBR322 at the
restriction boundary, and there is a frame-shift. Unlike Cases 2
and 3 before, this frame-shift splits the starting position of
the luciferase gene and completely destroys the luciferase
protein sequence. The bases on the line marked "fire 61" above
are grouped into codons as follows:
fire 61 ttt gaa aaA TGG AAA TGG AAA AGG AGG AGA ATG TTG TAT ATG GCC CTC TGC CAT TCT ACC peptides F E K W K W K R R R M L Y M A L C H S TCase 5. Inserting the luciferase gene at the fourth restriction site within tetr (at base 1268 of pBR322) also leaves a single base hanging beyond the last codon on pBR322 (pbr-luc8.sq4). The consequence is the same as Case 4 just considered.
pBR 1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
pBR 61 ttgctaacgc agtcaggcac cgtgtATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
pBR 121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
pBR 181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
:
:
pBR 1141 GCTGATCGTC ACGGCGATTT ATGCCGCCTC GGCGAGCACA TGGAACGGGT TGGCATGGAT
pBR 1201 TGTAGGCGCC GCCCTATACC TTGTCTGCCT CCCCGCGTTG CGTCGCGGTG CATGGAGCCG
pBR 1261 GGCCACCT
fire 1 cgaagtccc taaacggtag aggaaaagtt
fire 61 tttgaaaaAT GGAAATGGAA AAGGAGGAGA ATGTTGTATA TGGCCCTCTG CCATTCTACC
fire 121 CCATTGAAGA AGGATCAGCT GGAATTCAGT TGCATAAGTA CATGCATCAA TATGCCAAAC
fire 181 TTGGAGCAAT TGCTTTTAGT AACGCCCTTA CTGGAGTTGA CATTTCTTAC CAAGAATACT
fire 241 TTGATATTAC ATGTCGTTTA GCTGAGGCCA TGAAAAACTT TGGTATGAAA CCGGAAGAAC
fire 301 ATATTGCTTT GTGCAGTGAA AATTGTGAAG AATTTTTCAT CCCTGTACTT GCTGGTCTTT
:
... continued ...
Case 6. Finally, let's examine insertion into the beta-lactamase site at base 4017 of pBR322. Because both the P1 and P2 promoters as well as the gene controlled by them, beta-lactamase, are on the other strand, we need to either read backwards or examine the complementary sequence instead. Here, we will examine the complementary sequence of pBR322, which is contained in pbr322.out as a result of running one of the gene analysis programs in the handout. The restriction site at base 4017 in the original sequence is near base 4361-4017=344 in the complementary sequence, where 4361 is the size of the pBR322 plasimd. Part of the recombined gene sequence is shown below (pbr-luc8.sq5).
pBR 1 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
pBR 61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
pBR 121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
pBR 181 gcttcaataa tattgaaaaa ggaagagtAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT
pBR 241 TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT
pBR 301 AAAAGATGCT GAAGATCAGT TGGGTGCACG AGTGGGTTAC AT
fire 1 cgaagtccc taaacggtag aggaaaagtt
fire 61 tttgaaaaAT GGAAATGGAA AAGGAGGAGA ATGTTGTATA TGGCCCTCTG CCATTCTACC
fire 121 CCATTGAAGA AGGATCAGCT GGAATTCAGT TGCATAAGTA CATGCATCAA TATGCCAAAC
fire 181 TTGGAGCAAT TGCTTTTAGT AACGCCCTTA CTGGAGTTGA CATTTCTTAC CAAGAATACT
fire 241 TTGATATTAC ATGTCGTTTA GCTGAGGCCA TGAAAAACTT TGGTATGAAA CCGGAAGAAC
fire 301 ATATTGCTTT GTGCAGTGAA AATTGTGAAG AATTTTTCAT CCCTGTACTT GCTGGTCTTT
:
... continued ...
This is similar to Case 2, where there are two bases hanging
beyond the last codon (marked by green color). There is again a
premature termination of the beta-lactamase gene and a fresh
start of the complete luciferase gene.
Similarly, we can also reverse the culturing process to select luciferase inserted within the beta-lactamase gene: first culture the transformed cells on a tetracycline dish first then make a replica on a penicillin dish.
In practice, one would run samples through electrophoresis gels at each critical stage to make sure that the samples are good. Typical samples are: the partial restriction enzyme digests of pBR322, the partial digests of the luciferase gene, ligation product, plasmid DNA purified from transformed, screened, and selected cells.
|
