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Team:UC-Santa Cruz/Project/Pumps
From 2013.igem.org
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. After isolating and sequencing these plasmids, we found them to contain an 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, we had another source for Halorhodopsin.
We began finding a new HR source by searching for every organism that contained the gene. After this search we discovered that one of these organisms, Halobacterium Salinarum (HS), was in a frozen stock close by our lab on campus. Fortunately, the codon usage of the wild-type HR for H. salinarum is very similar to 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 restriction sites that could be used for ligation into our plasmids. (see tags section)
Our second pump is KR2, a newly discovered Sodium pump from a lab in South Korea. The gene for KR2 was found in the genome of Krokinobacter Eikastus, an organism which proved quite difficult to obtain. Additionally, we found that the codons used in the K. eikastus gene were not suitable for C. crescentus. By entering the codon usage table for C. Cresentus into an excel sheet with the DNA sequence with KR2 we were able to find the rare codons used and edit them for optimization in C. crescentus. G-blocks were then designed to be assembled with the PSB1C3 standard plasmid backbone. A Gibson Assembly was then run 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-
Credit: kazusa.or.jp
|
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/
