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Team:UC-Santa Cruz/Project/Pumps
From 2013.igem.org
The goal of the ion pumps group was to select and obtain ion
pumps to be expressed in C.Cresentus. Selection of ion pumps were based on ion selectivity and
structure simplicity.
Two pumps were used for this project. The first choice was
the widely studied ion pump, Halorhodopsin (HR), an
inward pointing conductor. For the sodium pump we chose the newly discovered
KR2, an outward pointing conductor.
First, we searched the iGEM
catalog for parts for anything containing the coding sequence for HR. We found
three parts; BBaK559000, BBaK9001 and BBaK559010 and
ordered them. Upon desiging sequencing primers and
sequencing of these plasmids, we found them to containing incomplete versions
of the coding sequence for HR from N.pharaonis.
Another problem was that the sequence of the HR in the parts contained an extra
base which was not in agreement with the coding
sequence the team who made the part reported. Luckily, running in parallel to
analysis of the parts we had another way to obtain the DNA coding for HR.
The other attempt was a search for every organism that
contained the gene was performed. Soon after, we searched our campus for stocks
of these organisms. It was soon discovered that one of these organisms, Halobacterium
Salinarium (HS)
was in a frozen stock close by our lab on campus. Fortunately, it was
discovered that the codon usage of the wild-type HR
for this HS was very much alike that of C. Cresentus,
therefore codon optimization was not required.
Specific primers were then designed to amplify the HR gene out of the HS
genome. The primers contain cut sites in order for ligation into our plasmids.
(see tags section)
Since KR2 is a newly discovered Sodium pump in a lab in
South Korea, it appeared to be very difficult to acquire the DNA for this
particular pump. The organism, Krokinobacter Eikastus ,which contains the
KR2 gene, also proved quite difficult to obtain. Additionally, the codon usage for K.Eikastus was
very different from C.Cresentus. In order to remedy
this, we entered in the codon usage table for C. Cresentus on an excel sheet and created a function to find
out how many rare codons were in the original KR2
sequence along with how many rare codons were next to
each other.
Through the use of G-blocks, we specified a desired sequence to be transformed into C. Cresentus. G-blocks (codon optimized for C. Cresentus) were ordered and contained sites to fit between the prefix and suffix with the PSB1C3 backbone . A Gibson reaction was then performed with the G-Blocks and the plasmid backbone to create our submission to the registry.
Cloning of a Newly discovered Na pumping Halorhodopsin from marine flavobacterium Krokinobacter eikastus
We used a Gibson reation with IDT G Blocks and IGEM pSB1C3 to clone KR2 halorhodopsin into pSB1C3 BioBrick backbone. Results confirmed by Sanger sequencing.
Key Reference- A light-driven sodium ion pump in marine bacteria. Inoue et al. Nature Communications DOI: 10.1038/ncomms2689 April 9, 2013.
DNA Sequence KR2 Na pumping halorhodopsin-
ATGACACAAGAACTAGGGAATGCCAATTTCGAAAATTTCATTGGAGCTACAG
AAGGATTTTCTGAAATTGCTTATCAATTTACATCACATATCCTTACGTTAGGGTACGCAGTGATGCTTGCAGGATT
ACTATACTTTATCCTTACCATCAAAAATGTAGATAAAAAATTCCAAATGTCGAACATATTATCAGCTGTGGTAATG
GTATCGGCATTTTTGCTATTATATGCGCAGGCACAAAACTGGACATCCAGTTTTACCTTTAATGAAGAAGTAGGAA
GATATTTTTTAGATCCGAGTGGTGATCTATTTAATAACGGATATCGCTATCTTAACTGGCTCATCGATGTACCTAT
GCTTCTCTTTCAAATTCTATTTGTAGTAAGTTTAACTACTTCAAAATTTAGCTCTGTACGTAACCAATTCTGGTTT
TCTGGGGCAATGATGATTATTACTGGGTACATTGGACAGTTTTATGAGGTAAGTAACTTGACTGCCTTTTTAGTAT
GGGGAGCTATTTCATCTGCTTTTTTCTTCCATATTTTATGGGTTATGAAGAAGGTAATTAATGAAGGAAAAGAGGG
GATTTCCCCAGCAGGACAAAAAATACTTTCTAATATCTGGATCTTATTTTTAATATCATGGACTTTATATCCAGGA
GCTTACTTAATGCCATACCTTACTGGAGTAGACGGATTTTTATATAGTGAAGATGGCGTGATGGCTAGACAACTAG
TATACACTATTGCAGATGTAAGTTCTAAAGTTATCTATGGTGTATTATTAGGTAACCTAGCAATTACATTAAGTAA
AAACAAAGAGTTGGTTGAAGCAAATAGCTAA
Protein Sequence KR2 Na pumping halorhodopsin-
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS*
Length: 280
Molecular weight: 31.524 kDa
Isoelectric point: 4.96
The codon
Usage table for Caulobacter crescentus-
|
Amino Acid |
Codon |
Number/ Genome |
#/1000 |
Fraction |
|
Ala |
GCG |
57020 |
46.06 |
0.334895631 |
|
Ala |
GCA |
3967 |
3.2 |
0.023299386 |
|
Ala |
GCT |
10175 |
8.22 |
0.059760839 |
|
Ala |
GCC |
99100 |
80.04 |
0.582044144 |
|
Arg |
AGG |
2701 |
2.18 |
0.029839039 |
|
Arg |
AGA |
1059 |
0.86 |
0.011699201 |
|
Arg |
CGG |
19055 |
15.39 |
0.210508291 |
|
Arg |
CGA |
4608 |
3.72 |
0.05090644 |
|
Arg |
CGT |
10018 |
8.09 |
0.110672897 |
|
Arg |
CGC |
53078 |
42.87 |
0.586374131 |
|
Asn |
AAT |
5773 |
4.66 |
0.196547733 |
|
Asn |
AAC |
23599 |
19.06 |
0.803452267 |
|
Asp |
GAT |
16529 |
13.35 |
0.231087561 |
|
Asp |
GAC |
54998 |
44.42 |
0.768912439 |
|
Cys |
TGT |
1309 |
1.06 |
0.142732526 |
|
Cys |
TGC |
7862 |
6.35 |
0.857267474 |
|
End |
TGA |
1799 |
1.45 |
0.472550565 |
|
End |
TAG |
1247 |
1.01 |
0.327554505 |
|
End |
TAA |
761 |
0.61 |
0.19989493 |
|
Gln |
CAG |
34155 |
27.59 |
0.859829318 |
|
Gln |
CAA |
5568 |
4.5 |
0.140170682 |
|
Glu |
GAG |
47647 |
38.48 |
0.713289121 |
|
Glu |
GAA |
19152 |
15.47 |
0.286710879 |
|
Gly |
GGG |
13983 |
11.29 |
0.126510929 |
|
Gly |
GGA |
5358 |
4.33 |
0.048476404 |
|
Gly |
GGT |
11281 |
9.11 |
0.102064635 |
|
Gly |
GGC |
79906 |
64.54 |
0.722948031 |
|
His |
CAT |
6487 |
5.24 |
0.293343583 |
|
His |
CAC |
15627 |
12.62 |
0.706656417 |
|
Ile |
ATA |
790 |
0.64 |
0.014615555 |
|
Ile |
ATT |
3549 |
2.87 |
0.065658995 |
|
Ile |
ATC |
49713 |
40.15 |
0.91972545 |
|
Leu |
TTG |
8478 |
6.85 |
0.068407378 |
|
Leu |
TTA |
427 |
0.34 |
0.003445382 |
|
Leu |
CTG |
84529 |
68.27 |
0.68204851 |
|
Leu |
CTA |
1742 |
1.41 |
0.014055868 |
|
Leu |
CTT |
7698 |
6.22 |
0.062113706 |
|
Leu |
CTC |
21060 |
17.01 |
0.169929156 |
|
Lys |
AAG |
40138 |
32.42 |
0.924284991 |
|
Lys |
AAA |
3288 |
2.66 |
0.075715009 |
|
Met |
ATG |
26760 |
21.61 |
1 |
|
Phe |
TTT |
5212 |
4.21 |
0.119489213 |
|
Phe |
TTC |
38407 |
31.02 |
0.880510787 |
|
Pro |
CCG |
37265 |
30.1 |
0.552000474 |
|
Pro |
CCA |
2760 |
2.23 |
0.040883438 |
|
Pro |
CCT |
4338 |
3.5 |
0.064258099 |
|
Pro |
CCC |
23146 |
18.7 |
0.342857989 |
|
Ser |
AGT |
1687 |
1.36 |
0.026704447 |
|
Ser |
AGC |
20348 |
16.44 |
0.322099631 |
|
Ser |
TCG |
26206 |
21.17 |
0.41482912 |
|
Ser |
TCA |
2127 |
1.72 |
0.033669447 |
|
Ser |
TCT |
2144 |
1.73 |
0.03393855 |
|
Ser |
TCC |
10661 |
8.61 |
0.168758805 |
|
Thr |
ACG |
20611 |
16.65 |
0.318075896 |
|
Thr |
ACA |
1775 |
1.43 |
0.027392398 |
|
Thr |
ACT |
1768 |
1.43 |
0.027284372 |
|
Thr |
ACC |
40645 |
32.83 |
0.627247334 |
|
Trp |
TGG |
17312 |
13.98 |
1 |
|
Tyr |
TAT |
11882 |
9.6 |
0.459918715 |
|
Tyr |
TAC |
13953 |
11.27 |
0.540081285 |
|
Val |
GTG |
37323 |
30.15 |
0.397755611 |
|
Val |
GTA |
1517 |
1.23 |
0.016166848 |
|
Val |
GTT |
6714 |
5.42 |
0.07155189 |
|
Val |
GTC |
48280 |
39 |
0.514525652 |
Codon optimized sequence for KR2 Na pumping halorhodopsin
ATGACGCAAGAACTGGGGAATGCCAATTTCGAAAATTTCATCGGTGCGACCG
AAGGCTTTAGCGAAATCGCCTATCAATTTACGTCGCATATCCTCACGCTGGGGTACGCGGTGATGCTCGCCGGGCT
GCTCTACTTTATCCTGACCATCAAGAATGTCGATAAGAAGTTCCAAATGTCGAACATCCTCAGCGCGGTGGTCATG
GTGTCGGCCTTTCTGCTCCTGTATGCGCAGGCGCAAAACTGGACCTCCTCGTTTACCTTTAATGAAGAAGTCGGTC
GTTATTTTCTCGATCCGAGCGGTGATCTGTTTAATAACGGCTATCGCTATCTCAACTGGCTCATCGATGTGCCCAT
GCTGCTCTTTCAAATCCTGTTTGTCGTGTCGCTCACGACCAGCAAGTTTAGCTCGGTCCGTAACCAATTCTGGTTT
AGCGGGGCCATGATGATCATCACGGGGTACATCGGGCAGTTTTATGAGGTGTCGAACCTGACCGCCTTTCTCGTCT
GGGGTGCGATCAGCTCGGCCTTTTTCTTCCATATCCTGTGGGTGATGAAGAAGGTCATCAATGAAGGCAAGGAGGG
GATCTCCCCGGCGGGGCAAAAGATCCTCAGCAATATCTGGATCCTGTTTCTCATCTCGTGGACGCTGTATCCCGGT
GCCTACCTCATGCCGTACCTGACCGGCGTGGACGGGTTTCTCTATAGCGAAGATGGCGTGATGGCGCGCCAACTGG
TCTACACGATCGCCGATGTGTCGAGCAAGGTCATCTATGGTGTGCTCCTGGGTAACCTCGCGATCACCCTGTCGAA
GAACAAGGAGCTCGTCGAAGCCAATAGCTAA
Protein Sequence for codon
optimized KR2 Na pumping halorhodopsin-
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS*
Alignment of two protein sequences,
one wild type and one codon optimized for Caulobacter (to confirm we have the same protein)
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS
*
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS
*
BioBrick pSB1C3 sequence with KR2 inserted- 2913 bp.
TACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCC
TGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGG
TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC
AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAC
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC
CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT
CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG
AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT
AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCT
CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG
GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG
GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG
ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGC
TTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCAT
TACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTG
TTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATC
AGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGT
TTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAA
ATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTAT
TCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCA
CCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGC
CGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAG
GTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATC
CAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGA
TCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAAC
CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCT
TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG/ATGACGCAAGAACTGGGGAATGCCAATTTCGAA
AATTTCATCGGTGCGACCGAAGGCTTTAGCGAAATCGCCTATCAATTTACGTCGCATATCCTCACGCTGGGGTACG
CGGTGATGCTCGCCGGGCTGCTCTACTTTATCCTGACCATCAAGAATGTCGATAAGAAGTTCCAAATGTCGAACAT
CCTCAGCGCGGTGGTCATGGTGTCGGCCTTTCTGCTCCTGTATGCGCAGGCGCAAAACTGGACCTCCTCGTTTACC
TTTAATGAAGAAGTCGGTCGTTATTTTCTCGATCCGAGCGGTGATCTGTTTAATAACGGCTATCGCTATCTCAACT
GGCTCATCGATGTGCCCATGCTGCTCTTTCAAATCCTGTTTGTCGTGTCGCTCACGACCAGCAAGTTTAGCTCGGT
CCGTAACCAATTCTGGTTTAGCGGGGCCATGATGATCATCACGGGGTACATCGGGCAGTTTTATGAGGTGTCGAAC
CTGACCGCCTTTCTCGTCTGGGGTGCGATCAGCTCGGCCTTTTTCTTCCATATCCTGTGGGTGATGAAGAAGGTCA
TCAATGAAGGCAAGGAGGGGATCTCCCCGGCGGGGCAAAAGATCCTCAGCAATATCTGGATCCTGTTTCTCATCTC
GTGGACGCTGTATCCCGGTGCCTACCTCATGCCGTACCTGACCGGCGTGGACGGGTTTCTCTATAGCGAAGA
TGGCGTGATGGCGCGCCAACTGGTCTACACGATCGCCGATGTGTCGAGCAAGGTCATCTATGGTGTGCTCCTGGGT
AACCTCGCGATCACCCTGTCGAAGAACAAGGAGCTCGTCGAAGCCAATAGCTAA/
