Team:FHS Frederick MD/LOV Domain

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=LOV Domain=
=LOV Domain=
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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.
+
LOV stands for light oxygen voltage. It is a sensor protein that detects the presence of blue light (365 nm). 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 efficiently 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 choose to modify LOV as our anaerobic environment and growth indicator. We choose LOV over green fluorescent proteins (GFP) due to the fact that GFPs are completely dependant on molecular oxygen to glow. However, due to FMN, LOV cannot release green light (495 nm).  
-
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.
+
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 LOV domain's cysteine amino acid, FMN had nothing to bind to.  
-
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.
+
Following Drepper's model we replaced the cysteine amino acid at position 62 of the ''Bacillus subtilis''-derived LOV domain with an alanine:
-
 
+
-
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.
+
-
 
+
-
<pre>
+
-
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-
+
-
</pre>
+
-
 
+
-
Therefore, our fluorescent protein will consist of the following sequence:
+
<pre>
<pre>
Line 37: Line 16:
</pre>
</pre>
-
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:
+
This is where our models departed. Instead of optimizing our gene for ''E.coli'', we choose to optimize codon usage for ''S. oneidensis''. We used the online [http://www.jcat.de/ Java Codon Adaptation Tool] to create a DNA sequence which has been optimized for our use in the following ways:
-
* Optimized for Shewanella oneidensis codon usage.
+
* Optimized for ''S. oneidensis'' codon usage.
* Avoids use of the four 3A Assembly restriction enzymes: EcoRI, SpeI, XbaI, PstI.
* Avoids use of the four 3A Assembly restriction enzymes: EcoRI, SpeI, XbaI, PstI.
* Avoids internal prokaryotic ribosome binding sites.
* Avoids internal prokaryotic ribosome binding sites.
-
When using this parameters, JCat produces the following nucleic acid sequence:
+
When using these parameters, JCAT produces the following nucleic acid sequence:
<pre>
<pre>
Line 53: Line 32:
ATGTTCTGGAACGAATTAAACATCGATCCAATGGAAATCGAAGATAAAAC    350
ATGTTCTGGAACGAATTAAACATCGATCCAATGGAAATCGAAGATAAAAC    350
TTACTTCGTTGGTATCCAAAACGATATCACTAAACAAAAAGAATACGAAA    400
TTACTTCGTTGGTATCCAAAACGATATCACTAAACAAAAAGAATACGAAA    400
-
AATTATTAGAATAA
+
AATTATTAGAA
</pre>
</pre>
-
Finally, we added the TAA stop codon to the end of the sequence to ensure termination of the translation.
+
Finally, we added a 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.
We used the sequence alignment tool to confirm that this was in fact the correct gene. The result show an exact match.
Line 63: Line 42:
100.0% identity in 137 residues overlap; Score: 712.0; Gap frequency: 0.0%
100.0% identity in 137 residues overlap; Score: 712.0; Gap frequency: 0.0%
-
Engineered     1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
+
Intended     1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
-
Expected      1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
+
Translated  1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
-
                ************************************************************
+
              ************************************************************
-
Engineered   61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
+
Intended   61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
-
Expected      61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
+
Translated  61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
-
                ************************************************************
+
              ************************************************************
-
Engineered   121 GIQNDITKQKEYEKLLE
+
Intended   121 GIQNDITKQKEYEKLLE
-
Expected    121 GIQNDITKQKEYEKLLE
+
Translated 121 GIQNDITKQKEYEKLLE
 +
              *****************
</pre>
</pre>
-
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:
+
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:
<pre>
<pre>
Line 93: Line 73:
</pre>
</pre>
-
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.
+
We also added the following terminal restriction sites to allow our LOV gene to be comparable with the 3A assembly process.
<pre>
<pre>
Line 102: Line 82:
</pre>
</pre>
-
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.
+
To check over our work we also ran the sequence through the [[http://tools.neb.com/NEBcutter2/index.php NEB Cutter]] to ensure that the 3A assembly restriction sites are correctly located at the ends of the sequence and not elsewhere within the open reading frame. The results show that the restriction sites are present where required and not elsewhere.
-
The results show that the restriction sites are present where required and not elsewhere.
+
-
This is the gene we need to order from IDT:
+
This is the BioBrick-formatted, ''Shewanella''-optimized, gene we ordered from the sequencing company for insertion into the pSB1C3 plasmid:
<pre>
<pre>
Line 120: Line 99:
TAGCGGCCGC TGCAGGAAGA AAC
TAGCGGCCGC TGCAGGAAGA AAC
</pre>
</pre>
 +
{{:Team:FHS_Frederick_MD/Footer}}

Latest revision as of 01:28, 21 June 2014

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LOV Domain

LOV stands for light oxygen voltage. It is a sensor protein that detects the presence of blue light (365 nm). 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 efficiently emit green light (495 nm) due to FMN.

We choose to modify LOV as our anaerobic environment and growth indicator. We choose LOV over green fluorescent proteins (GFP) due to the fact that GFPs are completely dependant on molecular oxygen to glow. However, due to FMN, LOV cannot 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 LOV domain's cysteine amino acid, FMN had nothing to bind to.

Following Drepper's model we replaced the cysteine amino acid at position 62 of the Bacillus subtilis-derived LOV domain with an alanine: 

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

This is where our models departed. Instead of optimizing our gene for E.coli, we choose to optimize codon usage for S. oneidensis. We used the online [http://www.jcat.de/ Java Codon Adaptation Tool] to create a DNA sequence which has been optimized for our use in the following ways:

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

When using these parameters, JCAT produces the following nucleic acid sequence: 

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

Finally, we added a 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%

Intended     1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
Translated   1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK
               ************************************************************

Intended    61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
Translated  61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV
               ************************************************************

Intended   121 GIQNDITKQKEYEKLLE
Translated 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

We also added the following terminal restriction sites to allow our LOV gene to be comparable with the 3A assembly process. 

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

To check over our work we also ran the sequence through the http://tools.neb.com/NEBcutter2/index.php NEB Cutter to ensure that the 3A assembly restriction sites are correctly located at the ends of 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 BioBrick-formatted, Shewanella-optimized, gene we ordered from the sequencing company for insertion into the pSB1C3 plasmid: 

>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
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