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Re: [DIYbio] Re: Do plasmids introduced via electroporation replicate with subsequent cell divisions?

10:19 PM |

You're going to need either a really good polymerase or a lot of ligase.

-SG

On Sat, Sep 30, 2017 at 9:59 PM Cory J. Geesaman <cory@geesaman.com> wrote:

Is it possible to create a synthetic chromosome containing a compilation of all the plasmids you want for eukaryotic cells to get them to replicate?

On Monday, September 25, 2017 at 8:50:50 AM UTC-4, Ravasz wrote:
"Why does nobody use plasmids to stably create proteins in eukaryotic cells?"

Because unlike bacteria, eukaryotic cells have chromosomes and they can't handle plasmids too well. When an eukaryotic cell replicates it will first duplicate its genome using multiple replication origins on each chromosome and then segregate the new chromosomes into daughter cells evenly. Special care is taken that one and only one copy of each chromosome is distributed to each daughter cell. This is regulated by the centromere of the chromosome. Plasmids lack both of these structures: they do not have the multiple replication origins that chromosomes do, and they do not have a centromere. So they are not reliably replicated, and then they are not evenly distributed to the daughter cells, so after a few rounds of replication they will be lost.
Also plasmids only work for up to about 15kb length, bigger plasmids will not be maintained in bacteria where you need to prepare them before inserting them into eukaryotes. Unfortunately, both the centromeres and eukaryotic replication origins tend to be several megabases in length, so you cannot put them into plasmids as they are just too big. So plasmids are too small to be able to contain the appropriate machinery to stably express protein in eukaryotic cells.
Therefore in eukaryotes other strategies are used: artificial chromosomes which are much larger than a plasmid, integrating plasmids which fuse into a host chromosome, or specially engineered plasmids which borrow mechanics from viruses to overcome these limitations.

Mate
On 23 September 2017 at 18:59, Mac Davis <mac.t...@gmail.com> wrote:
Why does nobody use plasmids to stably create proteins in eukaryotic cells?
On Sep 23, 2017 9:10 AM, "Ravasz" <ravasz...@gmail.com> wrote:
Hi,

I'm a bit late to the party but I wanted to chime in as I did my PhD on designing self-replicating plasmids for humans and now I am trying to engineer algae as a hobby.

As the previous replies already correctly stated, there is a mayor difference between prokaryotes and eukaryotes. Algae is a blanket term that encompasses organisms from both these domains so depending on exactly what species you want to work with you will need to go on very different routes.

Bacteria, as said by others here before me, will usually take up any plasmid that has a reasonable size and a matching replication origin. Most bugs will then gradually lose whatever genes you put on that plasmid as they will only want to hang on the selection markers which are hopefully encoded on your plasmid, while they will gradually switch off any other genes that don't provide them a growth advantage. You need specifically engineered bacteria that do not do that and keep expressing your genes of interest, but I'm afraid most popular alga species are notorious for having to be retransformed regularly as they just keep losing expression of any product they should be making.

For eukaryotes getting a plasmid to replicate in them is more tricky than it should be. It can be done but replication origins tend to be huge, complex and require several genes to work. I am happy to go into detail if interested, but by and large no one uses plasmids to stably express proteins in eukaryotes. Instead its a lot more popular to integrate the plasmid into the host genome, and this instantly solves the whole hassle of having to maintain an extra plasmid. So if eukaryotic algae are your thing, then you might want to use integrating plasmids for stable expression of any product you might need. Crispr-Cas9 or TALENs are also an option but these are more useful if you want to modify already existing genes rather than putting in something completely new.

If you feel like giving a bit more detail on what species you plan on using and what you want to achieve with it, then I think we can give you more specific information.

Cheers,
Mate

On Tuesday, 19 September 2017 17:33:09 UTC+1, Cory J. Geesaman wrote:
The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?

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Re: [DIYbio] Do proteins fold differently when folded by different cells?

10:17 PM |

Think about this: based on the contents of different solutions, more/less salt or higher/lower pH, proteins will be either folded (referred to as the proper conformational shape or conformation) or unfolded / denatured. This is because while proteins fold due to internal molecular interactions like hydrogen bonds, a large portion of protein folding has to do with the hydrophobic nature of different amino groups. As the solution changes, so do the hydration shells around the polypeptides and each cell is going to have its own unique internal chemistry.

When it comes to your question it's important to remember that it isn't as simple as just inserting DNA to a cell to get the protein created. Polypeptide chains are often processed through the endoplasmic reticulum, or the Golgi, where they pick up and lose things like disulfide bonds, cell pathway signals, or just help folding to find stable hydrogen bonds.

Protein signaling is pretty complex stuff, and studying the processing of proteins is difficult work. But, it's important to remember that bacteria have been making all the medical insulin on the planet since before either of us was born - so it can be done.

-SG

On Sat, Sep 30, 2017 at 9:52 PM Cory J. Geesaman <cory@geesaman.com> wrote:

Not necessarily different cells within a single organism, but say you were to splice a really large protein from a Human into a bacteria or the other way around (or any animal or plant or fungus, etc) - when the cell goes to synthesize that protein would it be likely to fold differently? "Likely" meaning "will it fold differently within that cell type more often than it might misfold otherwise."

If there is a difference in the way proteins are folded between different types of cells, is there any relatively comprehensive list of the differences and members of different groups of protein folding/misfolding mechanics?

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Re: [DIYbio] Re: Do plasmids introduced via electroporation replicate with subsequent cell divisions?

9:59 PM |

Is it possible to create a synthetic chromosome containing a compilation of all the plasmids you want for eukaryotic cells to get them to replicate?

On Monday, September 25, 2017 at 8:50:50 AM UTC-4, Ravasz wrote:

"Why does nobody use plasmids to stably create proteins in eukaryotic cells?"

Because unlike bacteria, eukaryotic cells have chromosomes and they can't handle plasmids too well. When an eukaryotic cell replicates it will first duplicate its genome using multiple replication origins on each chromosome and then segregate the new chromosomes into daughter cells evenly. Special care is taken that one and only one copy of each chromosome is distributed to each daughter cell. This is regulated by the centromere of the chromosome. Plasmids lack both of these structures: they do not have the multiple replication origins that chromosomes do, and they do not have a centromere. So they are not reliably replicated, and then they are not evenly distributed to the daughter cells, so after a few rounds of replication they will be lost.
Also plasmids only work for up to about 15kb length, bigger plasmids will not be maintained in bacteria where you need to prepare them before inserting them into eukaryotes. Unfortunately, both the centromeres and eukaryotic replication origins tend to be several megabases in length, so you cannot put them into plasmids as they are just too big. So plasmids are too small to be able to contain the appropriate machinery to stably express protein in eukaryotic cells.
Therefore in eukaryotes other strategies are used: artificial chromosomes which are much larger than a plasmid, integrating plasmids which fuse into a host chromosome, or specially engineered plasmids which borrow mechanics from viruses to overcome these limitations.

Mate

On 23 September 2017 at 18:59, Mac Davis <mac.t...@gmail.com> wrote:
Why does nobody use plasmids to stably create proteins in eukaryotic cells?

On Sep 23, 2017 9:10 AM, "Ravasz" <ravasz...@gmail.com> wrote:
Hi,

I'm a bit late to the party but I wanted to chime in as I did my PhD on designing self-replicating plasmids for humans and now I am trying to engineer algae as a hobby.

As the previous replies already correctly stated, there is a mayor difference between prokaryotes and eukaryotes. Algae is a blanket term that encompasses organisms from both these domains so depending on exactly what species you want to work with you will need to go on very different routes.

Bacteria, as said by others here before me, will usually take up any plasmid that has a reasonable size and a matching replication origin. Most bugs will then gradually lose whatever genes you put on that plasmid as they will only want to hang on the selection markers which are hopefully encoded on your plasmid, while they will gradually switch off any other genes that don't provide them a growth advantage. You need specifically engineered bacteria that do not do that and keep expressing your genes of interest, but I'm afraid most popular alga species are notorious for having to be retransformed regularly as they just keep losing expression of any product they should be making.

For eukaryotes getting a plasmid to replicate in them is more tricky than it should be. It can be done but replication origins tend to be huge, complex and require several genes to work. I am happy to go into detail if interested, but by and large no one uses plasmids to stably express proteins in eukaryotes. Instead its a lot more popular to integrate the plasmid into the host genome, and this instantly solves the whole hassle of having to maintain an extra plasmid. So if eukaryotic algae are your thing, then you might want to use integrating plasmids for stable expression of any product you might need. Crispr-Cas9 or TALENs are also an option but these are more useful if you want to modify already existing genes rather than putting in something completely new.

If you feel like giving a bit more detail on what species you plan on using and what you want to achieve with it, then I think we can give you more specific information.

Cheers,
Mate

On Tuesday, 19 September 2017 17:33:09 UTC+1, Cory J. Geesaman wrote:
The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?

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[DIYbio] Do proteins fold differently when folded by different cells?

9:52 PM |

Not necessarily different cells within a single organism, but say you were to splice a really large protein from a Human into a bacteria or the other way around (or any animal or plant or fungus, etc) - when the cell goes to synthesize that protein would it be likely to fold differently? "Likely" meaning "will it fold differently within that cell type more often than it might misfold otherwise."

If there is a difference in the way proteins are folded between different types of cells, is there any relatively comprehensive list of the differences and members of different groups of protein folding/misfolding mechanics?

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Re: [DIYbio] Re: Agilent 2100 Bioanalyzer Issues

11:15 AM |

I am running both RNA and DNA chips at different intervals and using a different cartridge for each one. This is why I concluded that it doesn't seem to be the cartridge itself, but could perhaps be an issue with cleaning (not drying beforehand, not rinsing for long enough, etc.), because we have been seeing the same issues for both RNA and DNA.

I haven't kept track of how closely the instances occur to the prep of new gel dye, but I'll be sure to look out for that in the future. Most times new gel is prepared almost weekly because we use this machine every day, but it's good to note.

The document you sent over is the standard protocol we have adapted, and we have even made a few further modifications ourselves. We vortex at just under 2000 RPM to ensure no splashing, and cleaning (both standard rinse and deep cleaning) is done on a regular basis. My current thought is that the diodes aren't fully drying, and that is what's causing voltage/migration issues. I'll be sure to let you know if that fixes the issue.

Thank you for the help.

-SG

On Thu, Sep 28, 2017 at 12:24 AM, Tobias Viehböck <tobi92.v@gmail.com> wrote:

Hey,
I also had problems during my first run, I even run expired chips. Are you running DNA or RNA chips? Does the problem also occur if you prepare the gel fresh?
This troubleshooting guide might be also useful https://biosci-batzerlab.biology.lsu.edu/Genomics/documentation/Mastering_the_Bioanalyzer_DNA-HS_assay.docx
although I modified it, e.g. I do all the pipetting with filter tips.

Hope that helps!

Am Montag, 25. September 2017 22:06:43 UTC+2 schrieb Skyler Gordon:
Hello all,

I'm not sure if anyone out there has any experience with this particular machine, but I've been having some issues with an Agilent 2100 Bioanalyzer. The machine is functioning very strangely, and I'm hoping someone might be able to help me figure out why.

Here's some background:
The machine has been serviced recently
The cartridge is relatively new (less than 1 year old)
The chips are not expired

Here is the issue:
When running samples on a chip, some will not read correctly. Extremely high fluorescence peaks, incorrect concentrations, and even the ladder will be incorrectly read on a regular basis. I have run some diagnostics using just ladder and just marker/reporter, and have determined that the machine will not malfunction on a consistent basis. This leads me to believe that the diodes within the cartridge are not damaged or functioning incorrectly.

Here's the rub:
When the machine does malfunction, I have had good success with just re-running the machine. I DO NOT change my samples, AT ALL. The chip, cartridge, software, etc. will all remain exactly the same. I do not even open the machine, I simply press start again and it tends to run successfully after a few runs. But the same issue occurs on subsequent runs, but not consistently across the different samples (i.e. sample well 2 will be incorrect on the initial run, but sample well 5 will be incorrect on the secondary run).

Again, not sure if there is anyone out there who has dealt with this, but I'm starting to get annoyed with this machine and I REALLY don't want to have to replace it.

-SG

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[DIYbio] Re: Agilent 2100 Bioanalyzer Issues

12:24 AM |

Hey,
I also had problems during my first run, I even run expired chips. Are you running DNA or RNA chips? Does the problem also occur if you prepare the gel fresh?
This troubleshooting guide might be also useful https://biosci-batzerlab.biology.lsu.edu/Genomics/documentation/Mastering_the_Bioanalyzer_DNA-HS_assay.docx
although I modified it, e.g. I do all the pipetting with filter tips.

Hope that helps!

Am Montag, 25. September 2017 22:06:43 UTC+2 schrieb Skyler Gordon:

Hello all,

I'm not sure if anyone out there has any experience with this particular machine, but I've been having some issues with an Agilent 2100 Bioanalyzer. The machine is functioning very strangely, and I'm hoping someone might be able to help me figure out why.

Here's some background:
The machine has been serviced recently
The cartridge is relatively new (less than 1 year old)
The chips are not expired

Here is the issue:
When running samples on a chip, some will not read correctly. Extremely high fluorescence peaks, incorrect concentrations, and even the ladder will be incorrectly read on a regular basis. I have run some diagnostics using just ladder and just marker/reporter, and have determined that the machine will not malfunction on a consistent basis. This leads me to believe that the diodes within the cartridge are not damaged or functioning incorrectly.

Here's the rub:
When the machine does malfunction, I have had good success with just re-running the machine. I DO NOT change my samples, AT ALL. The chip, cartridge, software, etc. will all remain exactly the same. I do not even open the machine, I simply press start again and it tends to run successfully after a few runs. But the same issue occurs on subsequent runs, but not consistently across the different samples (i.e. sample well 2 will be incorrect on the initial run, but sample well 5 will be incorrect on the secondary run).

Again, not sure if there is anyone out there who has dealt with this, but I'm starting to get annoyed with this machine and I REALLY don't want to have to replace it.

-SG

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Re: [DIYbio] Shouldn't we make a wiki?

9:52 AM |

On Wed, Sep 27, 2017 at 11:32 AM, Cathal Garvey <cathalgarvey@cathalgarvey.me> wrote:

HPlus has always been a communal effort AFAIK, though you might need to use IRC to get involved beyond merely contributing content.

That community has been around for almost 10 years now, take a look at http://diyhpl.us/wiki/hplusroadmap for a broad overview and about the IRC aspect, and also for contributing there's https://github.com/kanure/diyhpluswiki which has the same git repository. Originally there was a mailing list which began in 2007. The IRC logs http://gnusha.org/logs/ only go back to 2008. There's a lot of weird history in the IRC logs, like that time in January 2009 where I was commenting on some "lousy work" from "some guy Satoshi Nakamoto" .... although perhaps that oversight https://twitter.com/petertoddbtc/status/776900184836046849 is forgivable due to other circumstances.

There's some interesting resources on the wiki that you might be interested in; they make up mostly notes I guess:

http://diyhpl.us/wiki/gene-editing/

http://diyhpl.us/wiki/genetic-modifications/

one sentence summaries of all iGEM projects

http://diyhpl.us/wiki/dna/projects/

whole bunch of word-for-word transcripts from relevant conferences/meetings

http://diyhpl.us/wiki/transcripts/

IMHO the way to add content is to bookmark and link to diybio mailing list content, and just turn that into more pages. There's already a wealth of content and knowledge from this community, it's just a bit scattered. So if you want to spend 12 hours organizing the ol' mailbox file http://diyhpl.us/~bryan/irc/diybio.maildir.tar.gz then that's certainly one way to make sense of things.

There's also this stuff:
http://diyhpl.us/~bryan/papers2/diybio/

On Monday, September 25, 2017 at 9:40:10 AM UTC-7, Bryan Bishop wrote:
On Mon, Sep 25, 2017 at 11:37 AM, Michael Flynn <mfly...@gmail.com> wrote:
One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

There's a few different wikis floating around already--

https://openwetware.org/wiki/Main_Page
http://diyhpl.us/wiki/
http://diyhpl.us/wiki/diybio/faq/

- Bryan
http://heybryan.org/
1 512 203 0507

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Re: [DIYbio] Shouldn't we make a wiki?

9:32 AM |

Bryan's suggestions contain most of the great information that's available under a free license, afaik- because of the implicit license, like wikipedia, one can import that data freely into a newer effort. That's why standards, technology, and license are crucial considerations. ;)

I think OWW requires moderator approval to join, which is effective against spam but makes participation hard. HPlus has always been a communal effort AFAIK, though you might need to use IRC to get involved beyond merely contributing content.

On 27 September 2017 15:47:38 GMT+01:00, Michael Flynn <mflynn210@gmail.com> wrote:

Multiple information sources is part of the problem. There needs to be a place for consensus. If wikipedia was spread out over 10 different sites it would be 1/10^6 as effective. I wish I had time to put this together.

On Monday, September 25, 2017 at 9:40:10 AM UTC-7, Bryan Bishop wrote:
On Mon, Sep 25, 2017 at 11:37 AM, Michael Flynn <mfly...@gmail.com> wrote:
One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

There's a few different wikis floating around already--

https://openwetware.org/wiki/Main_Page
http://diyhpl.us/wiki/
http://diyhpl.us/wiki/diybio/faq/

- Bryan
http://heybryan.org/
1 512 203 0507

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Re: [DIYbio] Shouldn't we make a wiki?

8:33 AM |

For me, the wiki was the perfect way to learn pretty much everything i now know about synbio / diy bio etc. since once you wrote about a topic, you got an idea of it. That's also pretty much how www.synbio.info happend. First, it was just a notebook for me and then Eddy from deskgen had the idea to make it public and that's what we did.

Everytime the wiki idea starts off in this forum it get's after a few posts into a "which tech should we use" discussion, open source, etc. but I think that's not a relevant discussion for starting such a thing.

Much more interesting I find the question "What content would you want to have on such a wiki?", from that you would need to figure out on what level you start writing your articles. Should it be for complete novices in the bio field, should it be for biologists that have never worked with DIY Bio, should it address the greater public on what's going on in the field?

What are you thoughts on this?

Virenfrei. www.avast.com

On 27 September 2017 at 16:47, Michael Flynn <mflynn210@gmail.com> wrote:

Multiple information sources is part of the problem. There needs to be a place for consensus. If wikipedia was spread out over 10 different sites it would be 1/10^6 as effective. I wish I had time to put this together.

On Monday, September 25, 2017 at 9:40:10 AM UTC-7, Bryan Bishop wrote:
On Mon, Sep 25, 2017 at 11:37 AM, Michael Flynn <mfly...@gmail.com> wrote:
One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

There's a few different wikis floating around already--

https://openwetware.org/wiki/Main_Page
http://diyhpl.us/wiki/
http://diyhpl.us/wiki/diybio/faq/

- Bryan
http://heybryan.org/
1 512 203 0507

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Re: [DIYbio] Shouldn't we make a wiki?

7:47 AM |

Multiple information sources is part of the problem. There needs to be a place for consensus. If wikipedia was spread out over 10 different sites it would be 1/10^6 as effective. I wish I had time to put this together.

On Monday, September 25, 2017 at 9:40:10 AM UTC-7, Bryan Bishop wrote:

On Mon, Sep 25, 2017 at 11:37 AM, Michael Flynn <mfly...@gmail.com> wrote:
One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

There's a few different wikis floating around already--

https://openwetware.org/wiki/Main_Page
http://diyhpl.us/wiki/
http://diyhpl.us/wiki/diybio/faq/

- Bryan
http://heybryan.org/
1 512 203 0507

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Re: [DIYbio] Re: Shouldn't we make a wiki?

3:36 AM |

An open wiki would be cool, though the value of a wiki is in democracy and resilience, so an open standard would be best. I've considered GitHub wiki's because at least the data is standard format and easy to migrate, but Wikimedia is the generally-most-loved wiki out there.

Proprietary wikis are to be avoided IMO: at best they end up a semicommercial silo, at worst a graveyard of good intentions. I consider "Proprietary Wiki" to be a tautology, given the overtones of democratic editing Wikis have come to be associated with.

On 27 September 2017 07:35:39 GMT+01:00, bostjan <bostjan@irnas.eu> wrote:

Hi Michael,

I had the same thoughts when I joined the community and so we started this overview of DIY labware: https://github.com/symbiolab/bio-labware/blob/master/000_bio-labware_overview.md

Would be nice to have something like this for procedures as well...

All the best, B

Dne ponedeljek, 25. september 2017 18.37.16 UTC+2 je oseba Michael Flynn napisala:
One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

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Re: [DIYbio] Shouldn't we make a wiki?

11:58 PM |

I am newbie and I would really NEED a wiki!

Sent: Wednesday, September 27, 2017 at 8:35 AM
From: bostjan <bostjan@irnas.eu>
To: DIYbio <diybio@googlegroups.com>
Subject: [DIYbio] Re: Shouldn't we make a wiki?

Hi Michael,

I had the same thoughts when I joined the community and so we started this overview of DIY labware: https://github.com/symbiolab/bio-labware/blob/master/000_bio-labware_overview.md

Would be nice to have something like this for procedures as well...

All the best, B

Dne ponedeljek, 25. september 2017 18.37.16 UTC+2 je oseba Michael Flynn napisala:

One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

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[DIYbio] Re: Shouldn't we make a wiki?

11:35 PM |

One thing that's notably missing from this community is a centralized knowledge repository like a wiki.

This wiki would contain things like a "ramp" of procedures, annotated with dollar costs, to take someone from 0 knowledge in bio to someone that could make contributions.

Anyone else agree that this is missing? Perhaps we can make a kickstarter.

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New Java Position || San Jose, CA

3:18 PM |

Dear Partner,

Hope you are doing great!!

Please go through the requirement and let me know if you are having any consultant for this position.

Please share me the profile asap as this is a very hot requirement.

Java Developer

San Jose, CA

6+ Months

Job Description:

1. Python

2. Java

3. Bug fixes

4. Data fix

5. System maintenance

6. Listening, Verbal and Written Communication

Thanks,

Arpit Arora

Pyramid Consulting, Inc.

Executive-Resourcing

Desk Phone: 415.943.9386 E: Arpit.arora@pyramidci.com Web: www.pyramidci.com

WE FIND HIDDEN TALENT

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Exclusive Opening || Data warehousing engineer ||

12:51 PM |

Dear Partner,

Hope you are doing great!!

Please go through the requirement and let me know if you are having any consultant for this position.

Please share me the profile asap as this is a very hot requirement.

Senior Data warehousing engineer

Sunnyvale, CA

6+ Months

8 + years exp

Teradata experience

strong Business Intelligence background

Hadoop experience is a plus

Cassandra experience preferred

data security related experience preferred

knowledge of vertica will be a plus

Thanks,

Arpit Arora

Pyramid Consulting, Inc.

Executive-Resourcing

Desk Phone: 415.943.9386 E: Arpit.arora@pyramidci.com Web: www.pyramidci.com

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