Looking for Chloroplast integration Vector

[Mega]

Hey,

I wonder if  somebody knows where to get a chloroplast integration vector (Just 500bp flanking sites and MCS needed. And Origin of repl. and selection gene)

I think there should be some available?
Universal vectors with high or medium protein yield would be great. But also one-species (e.g. tobacco) vectors would be OK.

Are there companies selling it? Or researchers to share some?


all the best,
 

[Mega]

I guess I will have to make my own. If I understood correctly, it seems quite doable:


http://www.plantphysiol.org/content/145/4/1129.full
From here, I need to make primers for the described integration target.
Add restriction sites.
Then I  isolate some genetic material from chloroplasts (dirty) and put it into a PCR.

Make primers for KanamycinR.

Then fuse   flankingsite - KanR - Lux - flankingsite into some plasmid.  Ready (?)

[Mega]

Here is even a plasmid map

http://www.google.com/patents?id=DZ7WAAAAEBAJ&pg=PA3&source=gbs_selected_pages&cad=3#v=onepage&q&f=false


(http://www.google.com/patents/US7803991)

[Nathan McCorkle]

You don't need an ORI for integration if you can assemble then amplify
with PCR only

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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Mega]

Found something interesting:

You don't need Ribosome binding sites with Chloroplasts, it seems.

http://www.ncbi.nlm.nih.gov/pubmed/9520261

[Andreas Sturm]

Well another issue...

http://htmlimg3.scribdassets.com/1sv520l9a8vroop/images/9-dd963ed91a.jpg

psbA seems to be a promotor , a gene and a terminator??

I need the sequence of  the 5' and 3' untranslated region. http://www.ncbi.nlm.nih.gov/nuccore/?term=psbA gives no results... How would you look for it??

[Cory Tobin]

> I need the sequence of the 5' and 3' untranslated region.
> http://www.ncbi.nlm.nih.gov/nuccore/?term=psbA gives no results... How would
> you look for it??

According to the arabidopsis genome browser, psbA doesn't have any
UTRs. For example, here is a gene (CYCD3;1) that has both 5' and 3'
UTRs
http://gbrowse.arabidopsis.org/cgi-bin/gbrowse/arabidopsis/?name=AT4G34160
The UTRs are shown in light blue, the exons in dark blue and the
introns as thin lines. Here is the view of psbA
http://gbrowse.arabidopsis.org/cgi-bin/gbrowse/arabidopsis/?name=ATCG00020
No UTRs or introns are shown. I don't know much about chloroplast
gene structure so I have no intuition about whether or not this is
correct, but according to the data on arabidopsis.org that particular
gene has no UTRs.

-cory

[Cory Tobin]

> No UTRs or introns are shown. I don't know much about chloroplast
> gene structure so I have no intuition about whether or not this is
> correct, but according to the data on arabidopsis.org that particular
> gene has no UTRs.

After a little more digging I am almost positive this is incorrect.
This paper for example,
http://pcp.oxfordjournals.org/content/42/10/1071.full
claims the 5' UTR for psbA starts 77 nucleotides upstream from the
ATG. But none of the databases I can find have any mention of the
psbA UTRs.

Which species are you planning on working with? I might be able to
dig UTR coordinates out of the literature and cross-reference those
coordinates with the chloroplast genome sequence of that species.

-cory

[Andreas Sturm]

Thank you very much. Have already found this:

http://www.ncbi.nlm.nih.gov/nuccore/HQ130725.1

The sequence of a chloroplast integration plasmid!!

And the page has a feature that colours you the wanted sequence (promotor, Gene, UTRs)!!!


I don't have to search each sequence independently, I just switch the gene of interest where the CDS is. That makes it much easier!!

-cory

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[Nathan McCorkle]

Hey Andreas, the paper cited in that plasmid sequence is actually
really interesting.

Check out this quote:
"
Most of the current approaches used in generating plant based
vaccines relied on agrobacterium mediated transformation, that
target the genes to nucleus. The impinging problems of nuclear
transformation associated with low expression levels, position
effects due to random gene integration, and safety due to environmental
dissemination of genes by pollen has hampered its
expediency for commercialization [26,28]. Chloroplast transformation
technology has offered immense relief in this regard by
generating enormous expression levels [29]. Chloroplast transcription
machinery is akin to the prokaryotic system. Therefore,
multiple genes can be processed thus, facilitating the expression
of polyvalent vaccines. Other significant attributes include targeted
gene integration that precludes position effects and the
containment of transgenes due to maternal inheritance [30]. More
importantly, the proteins expressed in plastids were found to be
stable in the gut and were efficiently transported to the circulatory
system [31]. Previous studies have demonstrated successful
expression of antigens against cholera [32], tetanus [33], anthrax
[34,35], plague [36], ameobiasis [30], canine parvovirus [37], HIV
[38] and malaria [39] in chloroplasts. These studies have opened
new avenues for development of effective plant based vaccines.
The present study explores the feasibility of PA(dIV) expression in
plant system by nuclear and chloroplast transformation. The study
also compares the protective efficacy of plant expressed PA(dIV)
with that of recombinant domain IV i.e.: rPA(dIV) derived from
expression in E. coli.
"

here is the PDF:
http://diyhpl.us/~nmz787/A%20plant%20based%20protective%20antigen%20%5bPA(dIV)%5d%20vaccine%20expressed%20in%20chloroplasts%20demonstrates%20protective%20immunity%20in%20mice%20against%20anthrax.pdf

They cite 9 references to regarding prior chloroplast vaccine
expression... which means there could be 9 more vectors waiting to be
dug up!

Here are the references:
[30] Chebolu S, Daniell H. Chloroplast-derived vaccine antigens and
biopharmaceuticals:
expression, folding, assembly and functionality. Curr Top Microbiol
Immunol 2009;332:33–54.
[31] Limaye A, Koya V, Samsam M, Daniell H. Receptor-mediated oral delivery of a
bioencapsulated green fluorescent protein expressed in transgenic chloroplasts
into the mouse circulatory system. FASEB J 2006;20:959–61.
[32] Daniell H, Lee SB, Panchal T, Wiebe PO. Expression of the native
cholera toxin
B subunit gene and assembly as functional oligomers in transgenic tobacco
chloroplasts. J Mol Biol 2001;311:1001–9.
[33] Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, et al. Expression
of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res
2003;31:1174–9.
[34] Watson J, Koya V, Leppla SH, Daniell H. Expression of Bacillus
anthracis protective
antigen in transgenic chloroplasts of tobacco, a non-food/feed crop. Vaccine
2004;22:4374–84.
[35] Aziz MA, Sikriwal D, Singh S, Jarugula S, Kumar PA, Bhatnagar R.
Transformation
of an edible crop with the pagA gene of Bacillus anthracis. FASEB J
2005;19:1501–3.
[36] Arlen PA, Singleton M, Adamovicz JJ, Ding Y, Davoodi-Semiromi A, Daniell H.
Effective plague vaccination via oral delivery of plant cells expressing F1-V
antigens in chloroplasts. Infect Immun 2008;76:3640–50.
[37] Molina A, Hervas-Stubbs S, Daniell H, Mingo-Castel AM, Veramendi
J. Highyield
expression of a viral peptide animal vaccine in transgenic tobacco
chloroplasts. Plant Biotechnol J 2004;2:141–53.
[38] Scotti N, Alagna F, Ferraiolo E, Formisano G, Sannino L,
Buonaguro L, et al.
High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco
chloroplasts. Planta 2009;229:1109–22.
[39] Davoodi-Semiromi A, Schreiber M, Nalapalli S, Verma D, Singh ND, Banks
RK, et al. Chloroplast-derived vaccine antigens confer dual immunity against
cholera and malaria by oral or injectable delivery. Plant Biotechnol J
2010;8:223–42.
[40] Aziz MA, Singh S, Anand KP, Bhatnagar R. Expression of protective antigen
in transgenic plants: a step towards edible vaccine against anthrax. Biochem
Biophys Res Commun 2002;299:345–51.

[Nathan McCorkle]

Ahh this is great too, definitely read the section "UNIVERSAL VECTOR
VERSUS SPECIES-SPECIFIC CHLOROPLAST VECTORS"

Chloroplast Vector Systems for Biotechnology Applications
Dheeraj Verma and Henry Daniell
http://www.plantphysiol.org/content/145/4/1129.full

[Nathan McCorkle]

It seems the pLD vector sequence isn't online, at least from my
searchings, but in this patent there is a detailed map
http://www.google.com/patents/US7294506?printsec=drawing#v=onepage&q&f=false


from this
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764311/pdf/nihms-150656.pdf
Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals:
Expression, Folding, Assembly and Functionality
S. Chebolu and H. Daniell

"The main reason for the high cost of pharmaceutical protein
production is purification of
recombinant proteins. Therefore, novel protein purification strategies
can be used that do not
require the use of expensive column chromatography. For instance, a
synthetic protein-based
polymer gene (GVGVP) 121 has been expressed in E. coli and a very high
expression level was
achieved such that polymer inclusion bodies were formed that occupied
nearly 90% of the cell
volume (Daniell et al. 1997; Guda et al. 2000). (GVGVP) 121 exhibits
inverse temperature
transition properties, making it soluble in water below room
temperature, but aggregates into
a more ordered, viscoelastic state, called a coacervate, at 37°C
(Daniell et al. 1997; Guda et al.
2000). The inverse temperature transition property makes purification
easier and less expensive
in aqueous solutions simply by raising the temperature. In addition,
this property makes
(GVGVP) 121 an ideal fusion protein for purification"


and here's a way to get the DNA into the plastid with just PEG
http://diyhpl.us/~nmz787/stable%20plastid%20transformation%20in%20PEG-treated%20protoplasts%20of%20nicotiana%20tabacum.pdf

and check out this slideshow with a bunch of good links
www.slideshare.net/TNAUgenomics/multigene-engineering-in-plants

[Dakota]

So many cool things to learn.  Thanks for linking all this and starting the thread, I'll be ignoring my task at hand so I can read more about chloroplast integration vectors now!  I might have to stop reading DIYbio threads or I'll never finish one project.

[Andreas Sturm]

Read of Mr. Daniell several times a day. He's producing chloroplast integration vectors " on the assembly line" (very quicky). Or he's the only one.

Sent him an email request about a plasmid, didn't answer.

On Fri, Sep 7, 2012 at 2:44 PM, Dakota <dko...@gmail.com> wrote:

So many cool things to learn.  Thanks for linking all this and starting the thread, I'll be ignoring my task at hand so I can read more about chloroplast integration vectors now!  I might have to stop reading DIYbio threads or I'll never finish one project.

--

[Andreas Sturm]

Well I think it's easier to use a gene gun rather than using  PEG with protoplasts. You'll need the enzymes cellulase and pektinase to make protoplasts and I immagine that handling protoplasts is also more difficult than a piece of a leave...



The plasmid I mentioned, http://www.ncbi.nlm.nih.gov/nuccore/HQ130724.1 , it just says  "homologuos recombination site". Aha. I would choose trnA - trnI because I read multiple times that expression is strong there. But if I have it printed out just like this, it will still integrate in an active region?

[Andreas Sturm]

Hi,

Chloroplast sequences have to be ~ 500 bp in lenght. trnI - trnA ( = tRNA Ile  -  tRNA Ala )  is said to be a strong integration site. But I had a look at them, and they both have just ~ 38 nucleotides??!!??  How can they integrate then??


The trnI-trnA (and genes before and next to) sequences can be found here
http://www.ncbi.nlm.nih.gov/nuccore/NC_001879.2?report=genbank&from=104553&to=105331
Nucleotide: 102761  - 109139 


Actually, it should look like this, but as said, the flanking sites are much too small...
http://www.biomedcentral.com/1471-2229/10/213/figure/F1


Thx

[Cathal Garvey]

I don't actually know the answer to this question, because I've never
worked with Chloroplasts.

However, in similar cases, where people generally work with 500bp+ for
things like poorly-mapped promoters or for homologous recombination,
it's often because:
A) They never bothered cutting the recombination sites thinner than they
have them, as there was no need
B) Efficiency is somewhat higher with hundreds of bp but still works at
lower numbers
C) Special cases; some sites are just easier to integrate into, perhaps
because they are often unwound and their base-pairs interrupted by
proteins zipping through; this could easily be true of constitutive
genes like tRNAs.

Of course, in this case, a lot depends on how much space there is
between trnI and trnA. Homologous recombination isn't a straightfoward
cut-and-paste, it's more of an interweave-then-snip (look up "holliday
junction" for a headache). The "snip" site may be within the trnX genes,
but the interweaving bits might be a lot longer. Are the integrating
sequences flanked in the plasmid with only 38 nucleotides? Because that
*is* impressively small.

--
www.indiebiotech.com
twitter.com/onetruecathal
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PGP Public Key: http://bit.ly/CathalGKey

[Nathan McCorkle]

Actually in one of the papers I posted a few days ago, the SLICE paper
(using red lambda recombination machinery expression in e.coli) there
was peak efficiency around 50bp with it trailing off all the way into
the 100s. So I'm not surprised, but I would certainly do some more
research.

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[Mega]

>Are the integrating  sequences flanked in the plasmid with only 38 nucleotides

No, they are more than 1000! (I would cut them down to 600 when synthesizing)

But it doesn't say the site of integration. That's very bad.... And it may be anything, unlikely being trnI/trnA.

[Mega]

I found the sequence of the plasmid flanking sites in the genome:

tRNA | trnV | tRNA - Val    ++++  Unknown ++++ CDS (hypothetical protein ++++ Unknown (2094 nucleotides!)  ++++ Ribosomal protein s7



So actually it would put insert  in the middle of the unknown. Will this give expression? I think in chloroplast there are no position effects, so no matter where the insert is, it will be expressed? Can I trust the plasmid from NCBI to work?




I found an origin of replication between trnI and trnA, so replacing the stuff between them would even be lethal to the chloroplast?

[Nathan McCorkle]

On Mon, Sep 10, 2012 at 1:21 PM, Andreas Sturm <masters...@gmail.com> wrote:
> Hi,
>
> Chloroplast sequences have to be ~ 500 bp in lenght. trnI - trnA ( = tRNA
> Ile - tRNA Ala ) is said to be a strong integration site. But I had a
> look at them, and they both have just ~ 38 nucleotides??!!?? How can they
> integrate then??

Can you post a link to the paper/source you saw this? I could imagine
that means it's a good site for incorporation due to selection

[Nathan McCorkle]

yes it does say the site of integration:
http://www.ncbi.nlm.nih.gov/nuccore/HQ130724.1

misc_feature 1..1234
"derived from tobacco plastid genome accession Z00044; homologous
recombination sequence"
1 ataatcaggc tcgaactgat gacttccacc acgtcaaggt gacactctac cgctgagtta
61 tatcccttcc ccgccccatc gagaaataga actgactaat cctaagtcaa agggtcgaga
121 aactcaacgc cactattctt gaacaacttg gagccgggcc ttcttttcgc actattacgg
181 atatgaaaat aatggtcaaa atcggattca attgtcaact gcccctatcg gaaataggat
241 tgactaccga ttccgaagga actggagtta catctctttt ccattcaaga gttcttatgc
301 gtttccacgc ccctttgaga ccccgaaaaa tggacaaatt ccttttctta ggaacacata
361 caagattcgt cactacaaaa aggataatgg taaccctacc attaactact tcatttatga
421 atttcatagt aatagaaata catgtcctac cgagacagaa tttggaactt gctatcctct
481 tgcctagcag gcaaagattt acctccgtgg aaaggatgat tcattcggat cgacatgaga
541 gtccaactac attgccagaa tccatgttgt atatttgaaa gaggttgacc tccttgcttc
601 tctcatggta cactcctctt cccgccgagc cccttttctc ctcggtccac agagacaaaa
661 tgtaggactg gtgccaacaa ttcatcagac tcactaagtc gggatcacta actaatacta
721 atctaatata atagtctaat atatctaata taatagaaaa tactaatata atagaaaaga
781 actgtctttt ctgtatactt tccccggttc cgttgctacc gcgggcttta cgcaatcgat
841 cggattagat agatatccct tcaacatagg tcatcgaaag gatctcggag acccaccaaa
901 gtacgaaagc caggatcttt cagaaaacgg attcctattc aaagagtgca taaccgcatg
961 gataagctca cactaacccg tcaatttggg atccaaattc gagattttcc ttgggaggta
1021 tcgggaagga tttggaatgg aataatatcg attcatacag aagaaaaggt tctctattga
1081 ttcaaacact gtacctaacc tatgggatag ggatcgagga aggggaaaaa ccgaagattt
1141 cacatggtac ttttatcaat ctgatttatt tcgtaccttt cgttcaatga gaaaatgggt
1201 caaattctac aggatcaaac ctatgggact taag

here is Z00044's sequence
http://www.ncbi.nlm.nih.gov/nuccore/Z00044

the alignment starts at 140101 and ends at 141334 (used LOCAL
ALIGNMENT - WATER from http://www.ebi.ac.uk/Tools/psa/), which the
Z00044 page defines as being in "Inverted repeat A (IRA): 130599 -
155939 (25341 bp)"

they also note this about that particular area:
CDS 140283..140678
/note="ORF131"
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="CAA77401.1"
/db_xref="GI:1223680"
/translation="MKIMVKIGFNCQLPLSEIGLTTDSEGTGVTSLFHSRVLMRFHAP
LRPRKMDKFLFLGTHTRFVTTKRIMVTLPLTTSFMNFIVIEIHVLPRQNLELAILLPS
RQRFTSVERMIHSDRHESPTTLPESMLYI"
CDS complement(140315..140527)
/note="ORF70B"
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="CAA77402.1"
/db_xref="GI:1223681"
/db_xref="GOA:Q37073"
/db_xref="UniProtKB/TrEMBL:Q37073"
/translation="MKFINEVVNGRVTIILFVVTNLVCVPKKRNLSIFRGLKGAWKRI
RTLEWKRDVTPVPSESVVNPISDRGS"

If you see, about 3kb upstream is trnA and trnI

[Andreas Sturm]

>Can you post a link to the paper/source you saw this? I could imagine
>that means it's a good site for incorporation due to selection

I opened it with Genome compiler, there I can see the  genes  abstracted as bricks.

The only thing I could do is making screenshots, though.

>If you see, about 3kb upstream is trnA and trnI

But that doesn't count as trnI/trnA integration (does it? No)

[Omri Drory]

Hi Mega, that's a good feedback - what would you like to do? export as genbank format? copy/paste DNA? 

[Andreas Sturm]

Hm, I guess a "print button" would be nice, which exports/prints the selected sequence... Maybe in a format like jpeg, then anyone who has a computer can see it ;)




next,  I can insert nptI or nptII gene. Has anyone an idea which one will work in chloroplasts? (I assume both will work?)




 

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[Omri Drory]

I would use screenshots for now - would think about a nice way to "print" designs for publications and other needs in the future.

[Omri Drory]

+ we are working on cloud sharing/hosting of projects - for now you can email the gcproj file (need Genome Compiler) or as text.

[Andreas Sturm]

Well, the good-old screenshot approach will do the job until then :)

To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/K23eKJh4xisJ.

[Nathan McCorkle]

On Tue, Sep 11, 2012 at 10:23 AM, Andreas Sturm
<masters...@gmail.com> wrote:
>>Can you post a link to the paper/source you saw this? I could imagine
>>that means it's a good site for incorporation due to selection
>
> I opened it with Genome compiler, there I can see the genes abstracted as
> bricks.
>
> The only thing I could do is making screenshots, though.
>

So genome compiler is telling you trnI and trnA are 'strong'
recombination sites?

>
>
>>If you see, about 3kb upstream is trnA and trnI
>
> But that doesn't count as trnI/trnA integration (does it? No)

No, but I was asking where you ever got the idea that trnA and trnI
are 'strong' sites of integration... unless there's a transposon or
some other protein machinery that gives those sequences higher
priority/security I don't see why they would be any better than the
recombination sites already chosen in the integration plasmid. Maybe
the cell /is/ more protective of the DNA around the tRNA genes, so if
recombination happens there is more chance of it getting repaired or
something.

But you really have to post a reference for us to know what you're talking about

>
>
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[Andreas Sturm]

No, but I was asking where you ever got the idea that trnA and trnI
are 'strong' sites of integration... unless there's a transposon or
some other protein machinery that gives those sequences higher
priority/security

But you really have to post a reference for us to know what you're talking about

Well, I did some (actually many hours!)  google-research about chloroplast integration and that stuff, and I think it was written in a scientific paper.
I can look it up again.

Or was it the universal chloroplast integration vector at trnI/trnA?

> I don't see why they would be any better than the
> recombination sites already chosen in the integration plasmid. Maybe
> the cell /is/ more protective of the DNA around the tRNA genes, so if
> recombination happens there is more chance of it getting repaired or
> something.

So basically you say, I can use the sequence of  integration as it is, and it will probably be expressed?

[Andreas Sturm]

http://www.plantphysiol.org/content/145/4/1129.full

To date, the most commonly used site of integration is the transcriptionally active intergenic region between the trnI-trnA genes, within the rrn operon, located in the IR regions of the chloroplast genome. The foreign gene expression levels obtained from genes integrated at this site are among the highest ever reported (De Cosa et al., 2001). It appears that this preferred site is unique and allows highly efficient transgene integration and expression.

Chloroplast vectors may also carry an origin of replication that facilitates replication of the plasmid inside the chloroplast, thereby increasing the template copy number for homologous recombination and consequently enhancing the probability of transgene integration

So actually, all I have to do is take the flanking sequences, add my (prrn promotor) Lux-KanamycinR construct and the oriA.

[Nathan McCorkle]

On Tue, Sep 11, 2012 at 3:58 PM, Andreas Sturm <masters...@gmail.com> wrote:
>> I don't see why they would be any better than the
>> recombination sites already chosen in the integration plasmid. Maybe
>> the cell /is/ more protective of the DNA around the tRNA genes, so if
>> recombination happens there is more chance of it getting repaired or
>> something.
>
>
> So basically you say, I can use the sequence of integration as it is, and
> it will probably be expressed?

The integration site shouldn't matter to get expression, unless there
are DNA binding proteins (or histone-like proteins) clogging up the
region so transcription can't initiate. Otherwise expression is
determined by promoters and enhancers.

[Andreas Sturm]

What about this??

Or does the Replication origin need the "Unknown regions" too for being 'expressed'? because I used them for flanking and the system will be disrupted. They both have 180 nucleotides.

[Nathan McCorkle]

On Tue, Sep 11, 2012 at 4:14 PM, Andreas Sturm <masters...@gmail.com> wrote:
> http://www.plantphysiol.org/content/145/4/1129.full
>
>> To date, the most commonly used site of integration is the
>> transcriptionally active intergenic region between the trnI-trnA genes,
>> within the rrn operon, located in the IR regions of the chloroplast genome.
>> The foreign gene expression levels obtained from genes integrated at this
>> site are among the highest ever reported (De Cosa et al., 2001). It appears
>> that this preferred site is unique and allows highly efficient transgene
>> integration and expression.
>>
>> Chloroplast vectors may also carry an origin of replication that
>> facilitates replication of the plasmid inside the chloroplast, thereby
>> increasing the template copy number for homologous recombination and
>> consequently enhancing the probability of transgene integration

That makes a bit more sense, this too helps understanding, but its
still black magic (unless its demystified in the references)
"
All other earlier attempts on Rubisco engineering at other integration
sites within the chloroplast genome were only partially successful.
Integration of transgenes between exons of trnA and trnI also
facilitates correct processing of foreign transcripts because of
processing of introns present within both flanking regions.
"

You don't need anything in between the flanking sequences that you
don't need in the plant. An e.coli ori is not needed in the plant, so
keep it on the other side of the flanking site in the plasmid, you
don't need it to integrate into the plant chloroplast.

You should have something like this I think for the integration
shuttle plasmid (shuttle means you shuttle it between species, e.coli
and plant in this case),
recombination_site_1...promoter...CDS...terminator...recombination_site_2...all_the_e.coli_ori_and_antibiotic_selection_junk

I am probably missing some enhancers or RBS sites or something, but
that's the general idea

[Nathan McCorkle]

I'm not sure what you're trying to show or ask with the two screenshots

[Nathan McCorkle]

On Tue, Sep 11, 2012 at 4:37 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> You should have something like this I think for the integration
> shuttle plasmid (shuttle means you shuttle it between species, e.coli
> and plant in this case),
> recombination_site_1...promoter...CDS...terminator...recombination_site_2...all_the_e.coli_ori_and_antibiotic_selection_junk
>

and actually unless you just want to add a selection CDS, you'd need
another CDS for your GFP or lux operon... remember polycistronic
sequences work in chloroplast, so you might be able to easily get away
with just one promoter and one terminator

[Mega]

Yeah, I've written the operon.

Prrn promoter - 5' UTR (RBS, etc) - Lux C - LuxD - LuxA-B-E- KanamycinR - 3' UTR

Now i just  need the best recombination sites available, for maximum expression kevels. and those may be trnI/trnA.

[Omri Drory]

HI Mega, the recombination sites are for Tabaco plant only? will you need to redesign it for Arabidopsis or other plants?

[Andreas Sturm]

Hi Omri,

Theoretically, it perfectly fits only in tobacco.

However, in other plants the chloroplast genes may be the same, with silent mutations. For recombination, you don't need 100% homology, but the more base pairs fitting you have the more recombination events you'll get.


Clearly speaking: When it has worked in tobacco, I look up the sequence of Arabiodopsis chloroplast, and if it is similar (it may even work with >50% homology?)  to tobacco cp,  I would just try it.

 

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[Andreas Sturm]

That's crazy...

http://www.ncbi.nlm.nih.gov/nuccore/AY943927.1


There are barely flanking sites... Just some nucleotides... And that works?

[Nathan McCorkle]

Looks like 1kb flanking sequences on either side of the restriction sites, definitely sufficient (seqs from lettuce)

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[Andreas Sturm]

Definitely too much. I'll cut away some bps to make it 600 bp flanking per side, that will save us 400+400 bp of synthesis cost. :)
And will work as well? Or will it give 500% more transformants, then I'll leave it??

[Andreas Sturm]

I could have a chloroplast integration plasmid by now, but there's a patent on the marker cassette and the lab I asked  is not allowed to share it. Great.

[Dan Wright]

US patent? If you could send us the patent and the plasmid info, the people on this list may be able to find a way around the patent. Idle curiosity is usually fair use.

[Andreas Sturm]

http://www.pnas.org/content/early/2010/03/18/0914423107

On email request they said they would send me one, but there's a patent on the selectable marker cassette (aada - streptomycin). I can ask the patent holder for a material transfer agreement. He didn't answer though.

[Mega]

Ah, he also added that they did a very strict MTA with the patent holder to get the plasmid. 

So even if the patent was invalid in Europe he may not be allowed to share it. 

[Cathal Garvey]

What's the marker, what does it do, and what can we replace it with?

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[Nathan McCorkle]

"Spectinomycin resistance is conferred by the chimeric selectable
marker gene aadA (38), which is driven by plastid expression signals
(Fig. 3 A and B)."

38 is this - http://www.ncbi.nlm.nih.gov/nuccore/3859840

which references:
"9. Chinault, A. C., Blakesley, V. A., Roessler, E., Willis, D. G.,
Smith, C. A., Cook, R. G. & Fenwick, R. G. (1986) Plasmid
15, 119-131."

http://www.ncbi.nlm.nih.gov/pubmed/3517903

full article here:
http://diyhpl.us/~nmz787/Characterization%20of%20Transferable%20Plasmids%20from%20Shigella%20flexneri%202a%20That%20Confer%20Resistance%20to%20Trimethoprim,%20Streptomycin,%20and%20Sulfonamides.pdf

Both NCBI links have an associated Nucleotide section on the lower
right-hand side of the page

[Andreas Sturm]

Cathal,
as I got no answer from the patent-holder I asked the lab whether they'd have one without the patented marker, be it marker-free or any other marker. Negative.


(
Anyway, I don't really like resistances other than kanamycin (but I'd have taken the plasmid for sure!)  ;)  The (unlikely) escape of kanamycin genes into nature wouldn't affect the environment as kR is wide-spread in most soil bacteria. Barely used therapeutically in humans any more today.
)

[Cathal Garvey]

Well, I don't know how useful they are in plant engineering, but I've
looked at medically unimportent *bacteriocins* in the past as potential
selective agents. There are loads of bacteriocins (peptide antibiotics)
that are unused because they might trigger allergies, and because they
can't be orally taken as medicines (peptides get digested!).
Lantibiotics are another avenue here; some of them are medically
interesting, but others are likely to be dead-ends, and might be
penetrative enough to use with plants if they affect chloroplasts.

That's the biggie though; do they affect chloroplasts? I don't think
anyone tests Bacteriocins for that sort of thing! Perhaps look through
the bacteriocin databases for ones known to affect species that are
affected by kanamycin, and work from there; do they affect cell wall
synthesis? Then check if chloroplasts have similar cell walls to the
affected species. Do they affect protein translation? Compare the
chloroplast ribosomes to affected species and make a guess.

Wild west territory, this is. :)

[Andreas Sturm]

Actually that's what should have been done by labs that recieve government funding, developing selection agents that are useless for human therapy.
Wouldn't have been a big deal if many people worked on it...


Here's a gene (the only one?) that codes for an lantibiotic, nisin.... nsuA

And that's the protection against nisin..
http://www.ncbi.nlm.nih.gov/nuccore/NC_015600.1?report=genbank&from=1144887&to=1147862&strand=true

in the Illustration, http://www.ncbi.nlm.nih.gov/gene/10752676 , it seems that it's an entire operon... nsuEGAB...



Would be great, if the bacteria would themselfes kill all the other bacteria which have lost the plasmid :) (As long as the plasmid just codes for GFP, Lux, medical substances or something harmless)



After all, with the glowing plant I think I have to stay by what is already commonly used in "main-stream science" ;)  Kanamycin was shown to work and be the most harmless of antibiotics. Developing an apropriate lantibiotic operon will consume time and make the project even more complex... Maybe there's an IGEM team to work on that :)

[Mega]

Another question ;)


What if I clone in the lux operon twice?

Saying: Promoter - 5' UTR - LuxCDABE-CDABE-KanamycR - 3'UTR

Will it be twice as bright??


It will then be ~ 20 kbp (or a bit smaller). Can you still amplify it in E.coli? Or is it too big to enter cells by heat shock?
And will it get inside the plant cells when Polyethylenglycol transformation of protoplasts or gene gun is applied?


Tx

[Cathal Garvey]

I looked into this for my own plasmid; there are closely related operons
to produce things very like Nisin, which are toxic to B.subtilis strains
not carrying the immunity gene. The only reason I didn't include them at
the time was that they'd have tripled the cost of my plasmid!

That's the primary reason nobody's bothered lately, I think. An
antibiotic resistance gene like beta-lactamase (AmpR) is one reasonably
small gene. It has lots of disadvantages, like conferring community
resistance (so there's always a significant subset of non-resistant
cells), but it's small. This means that plasmids that aren't stable with
large inserts can carry it and still have room for other stuff.

But, if you have a plasmid that *can* carry large loads, a more
complicated system is awesome for enforcing peer-monitoring among your
bacteria. Call it the "Stasi Operon". :P

Nisin is one of the useful bacteriocins though; it's used as a food
preservative. But, there are plenty of ones that are very similar,
sometimes with smaller operons. I can't recall the databases, and I'm
sending this while offline so I can't check, but there are at least two
decent databases out there for bacteriocins and lantibiotics that list
known susceptable strains, natural producers, and link to the operons,
etc.. Just be prepared for lots of DNA, because making antibiotics is
always costly.

[xmort]

I am just guessing that your goal is high expression of lux. If this is the case, I am afraid you are not going to get any higher expression by doubling the genes. There are hundreds of copies of chloroplast genome per each chloroplast and hundreds or even thousands chloroplasts per cell. So in most cases your expression level is not limited by low gene load or inefficient transcription, but by the protein stability and related issues.
The double casette also considerable increases the probability of homologous recombination. Basically the same process you are trying to take advantage of to integrate your DNA into chloroplast genome is going to cut out better part of your gene of interest.
 

[Andreas Sturm]

Ah, thank you! haven't considered that HR will cut it out sooner or later.

What about introducing Red fluorescent protein, will it absorb 1 photon of the blueish-green light and make 5 photons of red light out of it  (better visibility)? (Just some imaginary numbers)

On Mon, Oct 1, 2012 at 3:53 PM, xmort <mor...@ueb.cas.cz> wrote:

I am just guessing that your goal is high expression of lux. If this is the case, I am afraid you are not going to get any higher expression by doubling the genes. There are hundreds of copies of chloroplast genome per each chloroplast and hundreds or even thousands chloroplasts per cell. So in most cases your expression level is not limited by low gene load or inefficient transcription, but by the protein stability and related issues.
The double casette also considerable increases the probability of homologous recombination. Basically the same process you are trying to take advantage of to integrate your DNA into chloroplast genome is going to cut out better part of your gene of interest.

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