LOV Domain

LOV stands for light oxygen voltage. It is a sensor protein that detects the presence of blue light(365nm). In its wild type form it is used by higher plants, fungi, and bacteria. In higher plants LOV controls phototropism and chloroplasts relocation. In this form it absorbs blue light (365 nm) and in the wild state flavin mononucleotides (FMN) link to cysteine. This results in LOV not being able to emit green light (495 nm) due to FMN. We choose to modify LOV as are anaerobic environment and growth indicator. We choose love over green fluorescent proteins (GFP) due to the fact that GFPs are completely depend on molecular oxygen to glow. However due to FMN LOV can not release green light (495 nm). Thomas Drepper found a solution to this problem Drepper and his fellow researchers realized the effects of FMN and found away to remove it. By eliminating the cysteine amino acid FMN had nothing to bind to. Following Dreppers model we removed the cysteine amino acid from bacillus subtilis.

To create the LOV strand, we initially created cultures of E.coli containing the plasmid pBB1MCS2. We chose this plasmid due to its ability to transform a wide range of bacteria, including our desired host bacterial strain of Schwenella odeneidensis for the LOV gene. We grew it in kanamycin-rich plates to eliminate all possible bacterial colonies lacking pBB1MCS2. Using the Quia prep spin mini prepkit, we extracted the plasmid from the E.coli bacteria. We tested the purity of the uncut plasmid through electrophoresis.

We then grew cultures of Bacillus subtilis that contained the desired LOV domian. Extraction of the genomic DNA from B.subtilis was accomplished through a boil prep procedure.

We then digested LOV and NirB into two separate preps containing the plasmid psB1C3 already treated with EcoRI and PstI,which originated from the iGEM 3A assembly kit.

Ligation of the two digests of NirB and LOV were completed fusing them to the plasmid psB1C3. Using these new plasmids, we then commenced on transformation of the LyoComp cells. However the transformation efficiency was very low.. After two failed transformations, we decided to create our own chemically competent cells,thus created E.coli NE1U beta cells. We then performed another digestion, as well as ligation, with LOV and NirB. We completed another transformation using E.coli NE1U Beta bacteria with the prepared plasmids. Following the incubation period, we saw growth on all plates cultured,with the exception of the negative control. Thus we can conclude that we extracted the two plasmids using the mini prep kit and successfully transformed those plasmids into the bacteria to show expression.

LOV Domain

LOV stands for light oxygen voltage. It is a sensor protein that detects the presence of blue light(365nm). In its wild type form it is used by higher plants, fungi , and bacteria. In higher plants LOV controls phototropism and chloroplasts relocation. In this form it absorbs blue light(365nm) and in the wild state flavin mononuclotides(FMN) link to cysteine. This results in LOV not being able to emit green light(495nm) due to FMN.

We choose to modify LOV as are anaerobic environment and growth indicator. We choose love over green fluorescent protiens(GFP) due to the fact that GFPs are completely depend on molecular oxygen to glow. However due to FMN LOV can not release green light(495nm). Thomas Drepper found a solution to this problem

Drepper and his fellow researchers realised the effects of FMN and found away to remove it. By eliminating the cysteine amino acid FMN had nothing to bind to. Following Drappers model we removed the cysteine amino acid from bacillus subtilis.

001 MASFQSFGIP GQLEVIKKAL DHVRVGVVIT DPALEDNPIV YVNQGFVQMT GYETEEILGK
061 NCRFLQGKHT DPAEVDNIRT ALQNKEPVTV QIQNYKKDGT MFWNELNIDP MEIEDKTYFV
121 GIQNDITKQK EYEKLLEDSL TEITALSTPI VPIRNGISAL PLVGNLTEER FNSIVCTLTN
181 ILSTSKDDYL IIDLSGLAQV NEQTADQIFK LSHLLKLTGT ELIITGIKPE LAMKMNKLDA
241 NFSSLKTYSN VKDAVKVLPI M-

Therefore, our fluorescent protein will consist of the following sequence:

001 MASFQSFGIP GQLEVIKKAL DHVRVGVVIT DPALEDNPIV YVNQGFVQMT GYETEEILGK
061 NARFLQGKHT DPAEVDNIRT ALQNKEPVTV QIQNYKKDGT MFWNELNIDP MEIEDKTYFV
121 GIQNDITKQK

This is where are models departed. Instead of optimizing are gene for E.coli. we choose to to optimize codon usage for Shewanella Onedensis. Throughout the use the online Java Codon Adaptation Tool (http://www.jcat.de/) to create a DNA sequence which has been optomized for our use in the following ways:

• Optimized for Shewanella oneidensis codon usage.
• Avoids use of the four 3A Assembly restriction enzymes: EcoRI, SpeI, XbaI, PstI.
• Avoids internal prokaryotic ribosome binding sites.

When using this parameters, JCat produces the following nucleic acid sequence:

ATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTCAATTAGAAGTTATCAA     50
AAAAGCTTTAGATCACGTTCGTGTTGGTGTTGTTATCACTGATCCAGCTT     100
TAGAAGATAACCCAATCGTTTACGTTAACCAAGGTTTCGTTCAAATGACT     150
GGTTACGAAACTGAAGAAATCTTAGGTAAAAACGCTCGTTTCTTACAAGG     200
TAAACACACTGATCCAGCTGAAGTTGATAACATCCGTACTGCTTTACAAA     250
ACAAAGAACCAGTTACTGTTCAAATCCAAAACTACAAAAAAGATGGTACT     300
ATGTTCTGGAACGAATTAAACATCGATCCAATGGAAATCGAAGATAAAAC     350
TTACTTCGTTGGTATCCAAAACGATATCACTAAACAAAAAGAATACGAAA     400
AATTATTAGAATAA

Finally, we added the TAA stop codon to the end of the sequence to ensure termination of the translation.

We used the sequence alignment tool to confirm that this was in fact the correct gene. The result show an exact match.

100.0% identity in 137 residues overlap; Score: 712.0; Gap frequency: 0.0%

Engineered     1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
Expected       1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
                ************************************************************

Engineered    61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
Expected      61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
                ************************************************************

Engineered   121 GIQNDITKQKEYEKLLE
Expected     121 GIQNDITKQKEYEKLLE

We used an E. coli codon usage application to see how well it will do in the bacteria. This analysis shows that there are multiple occurrences of the codon TTA (for leucine) which is poorly expressed in E. coli. The next most common Leucine codon in Shewanella is CTG, which is also well tolerated in E. coli. The following sequence replaces every instance of the Leucine TTA codon with CTG:

atggcttctttccaatctttcggtatcccaggtcaactggaagttatcaaaaaagctctg
M  A  S  F  Q  S  F  G  I  P  G  Q  L  E  V  I  K  K  A  L
gatcacgttcgtgttggtgttgttatcactgatccagctctggaagataacccaatcgtt
D  H  V  R  V  G  V  V  I  T  D  P  A  L  E  D  N  P  I  V
tacgttaaccaaggtttcgttcaaatgactggttacgaaactgaagaaatcctgggtaaa
Y  V  N  Q  G  F  V  Q  M  T  G  Y  E  T  E  E  I  L  G  K
aacgctcgtttcctgcaaggtaaacacactgatccagctgaagttgataacatccgtact
N  A  R  F  L  Q  G  K  H  T  D  P  A  E  V  D  N  I  R  T
gctctgcaaaacaaagaaccagttactgttcaaatccaaaactacaaaaaagatggtact
A  L  Q  N  K  E  P  V  T  V  Q  I  Q  N  Y  K  K  D  G  T
atgttctggaacgaactgaacatcgatccaatggaaatcgaagataaaacttacttcgtt
M  F  W  N  E  L  N  I  D  P  M  E  I  E  D  K  T  Y  F  V
ggtatccaaaacgatatcactaaacaaaaagaatacgaaaaactgctggaataa

Using iTEMs recommendation we also added the following terminal restriction sites. To allow are LOV gene to be comparable with the three A assembly process.

 Prefix
5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG[part] 3'
Suffix
5' [part]TACTAGTAGCGGCCGCTGCAGGAAGAAAC 3'

To check over are work We will also ran the sequence the the NEB Cutter to ensure that the 3A assembly restriction sites are correctly located at the ends of the the sequence and not elsewhere within the open reading frame. The results show that the restriction sites are present where required and not elsewhere.

This is the gene we need to order from IDT:

>LOV
GTTTCTTCGA ATTCGCGGCC GCTTCTAGAG atggcttctt tccaatcttt 
cggtatccca ggtcaactgg aagttatcaa aaaagctctg gatcacgttc 
gtgttggtgt tgttatcact gatccagctc tggaagataa cccaatcgtt 
tacgttaacc aaggtttcgt tcaaatgact ggttacgaaa ctgaagaaat 
cctgggtaaa aacgctcgtt tcctgcaagg taaacacact gatccagctg 
aagttgataa catccgtact gctctgcaaa acaaagaacc agttactgtt 
caaatccaaa actacaaaaa agatggtact atgttctgga acgaactgaa 
catcgatcca atggaaatcg aagataaaac ttacttcgtt ggtatccaaa 
acgatatcac taaacaaaaa gaatacgaaa aactgctgga ataaTACTAG 
TAGCGGCCGC TGCAGGAAGA AAC