Team:UC-Santa Cruz/Project/Pumps

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Revision as of 03:29, 28 September 2013

Ion Pumps

The goal of the ion pumps group was to find and obtain light driven sodium and chloride pumps to be expressed in C. cresentus. Choice of ion pumps was based on ion selectivity and structure simplicity.

Two pumps were used for this project. The first choice was the widely studied Halorhodopsin (HR), an inward pointing chloride conductor. For the sodium pump we chose the newly discovered KR2, an outward facing pump.

First, we searched the iGEM catalog for any parts containing the coding sequence for HR. We found three parts; BBaK559000, BBaK9001 and BBaK559010 and requested them for use in our project. After isolating and sequencing these plasmids, we found them to contain 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. With this we searched for a new source to obtain the Halorhodopsin gene from.

Our next attempt was to search other organisms that contained the HR gene. After this we found 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 was very similar 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 for ligation into our plasmids. (see tags section)

Our second pump is KR2, a newly discovered light driven sodium pump from a lab in South Korea. The organism which contains the KR2 gene, Krokinobacter Eikastus, 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 with the KR2 DNA sequence and identified the rare codons.

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
*

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/

@@ Line 156: / Line 156: @@
 <p class=MsoNormal><span style='font-family:"Times New Roman";mso-fareast-font-family:
 "Times New Roman"'>Two pumps were used for this project. The first choice was
-the widely studied ion pump, <span class=SpellE>Halorhodopsin</span> (HR), an
+the widely studied <span class=SpellE>Halorhodopsin</span> (HR), an
-inward pointing conductor. For the sodium pump we chose the newly discovered
+inward pointing chloride conductor. For the sodium pump we chose the newly discovered
-KR2, an outward pointing conductor.<o:p></o:p></span></p>
+KR2, an outward facing pump.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:"Times New Roman";mso-fareast-font-family:
@@ Line 172: / Line 172: @@
 Another problem was that the sequence of the HR in the parts contained an extra
 <span class=GramE>base which</span> was not in agreement with the coding
-sequence the team who made the part reported. Luckily, we had another source to obtain the Halorhodopsin gene.<span
+sequence the team who made the part reported. With this we searched for a new
+source to obtain the Halorhodopsin gene from.<span
 style="mso-spacerun: yes">&nbsp;&nbsp; </span><o:p></o:p></span></p>
@@ Line 180: / Line 181: @@
 <p class=MsoNormal><span style='font-family:"Times New Roman";mso-fareast-font-family:
 "Times New Roman"'>Our next attempt was to search other organisms that
-contained the HR gene. Soon after, we discovered that one of these organisms<span
+contained the HR gene. After this we found that one of these organisms<span
 class=GramE>,<span style="mso-spacerun: yes">&nbsp; </span><span class=SpellE>Halobacterium</span></span>
 <span class=SpellE>Salinarum</span> (HS)<span style="mso-spacerun: yes">&nbsp;
@@ Line 193: / Line 194: @@
 <p class=MsoNormal><span style='font-family:"Times New Roman";mso-fareast-font-family:
-"Times New Roman"'>Since KR2 is a newly discovered Sodium pump in a lab in
+"Times New Roman"'>Our second pump is KR2, a newly discovered light driven sodium pump from a lab in
-South Korea, it appeared to be very difficult to acquire the DNA for this
+South Korea. The organism which</span> contains the KR2 gene, Krokinobacter <span class=SpellE><span
-particular pump. The organism, Krokinobacter <span class=SpellE><span
+class=GramE>Eikastus</span></span><span class=GramE>, proved quite difficult to obtain. Additionally, the <span
-class=GramE>Eikastus</span></span><span class=GramE> ,which</span> contains the
+class=SpellE>codon</span> usage for <span class=SpellE>K. eikastus</span> was
-KR2 gene, also proved quite difficult to obtain. Additionally, the <span
+very different from <span class=SpellE>C. cresentus</span>. In order to remedy
-class=SpellE>codon</span> usage for <span class=SpellE>K.Eikastus</span> was
-very different from <span class=SpellE>C.Cresentus</span>. In order to remedy
 this, we entered in the <span class=SpellE>codon</span> usage table for C. <span
-class=SpellE>Cresentus</span> on an excel sheet and created a function to find
+class=SpellE>cresentus</span> on an excel sheet with the KR2 DNA sequence and identified the
-out how many rare <span class=SpellE>codons</span> were in the original KR2
+rare <span class=SpellE>codons.</span></p>
-sequence along with how many rare <span class=SpellE>codons</span> were next to
-each other.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:"Times New Roman";mso-fareast-font-family: