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From 2011.igem.org

Action points for the day:
-bios on the wiki
-sci-fi meeting
-characterisation talk
-come up with more ideas
-do more research on anti-venom and prodigiosin


Sci-Fi prodigiosin ideas (developed with Nick Kral and CJ from the RCA):
-use prodigiosin (red pigment) as the new "colour of health" (know something is sterile rather than assume it is)
-possible future uses: in decontamination/ as a "panic room"/ sterile hospice
-possible future products: hand gel, clothing (e.g. protective suits in bioreactor plants), decontamination paint (in hospitals etc)
-actual uses: anti-cancer (could be in a red drip, red pill?), anti-malarial (drug verification because pigment colour is hard to fake)
Eventually, this idea was scratched because optimising the production pathway does not contain enough synthetic biology. In addition, the compound is immunosuppressive (http://pubs.rsc.org/en/Content/ArticleHtml/2008/CC/b719353j) and would therefore be disadvantageous to normal people. Many of the envisaged applications would only work with less problematic analogues of prodigiosin.

Brainstorming:
Venom and BAS ideas to be presented on Friday!
-desertification (overdone already)
-thermophiles (make them express melanin so they can be heated up by the sun, saving energy)
-cell-cell signal transduction (can be used as inhibition or activation)
-desalination (commercial membranes are probably more efficient)
-data storage in bacteria (use DNA to encode information has already been done but it might be useful to come up with a new way of retrieving the information. Storing data in RNA might be more efficient)
-ocean metals (retrieving valuable materials from sea water, main problem: materials are only present at really low concentration and might be hard to retrieve) -cross-linking of hair to something useful
-dictostylium
-using melanin for heat production from sound in bacteria. The heat could then be converted into an output, producing sound sensitive bacteria. Is melanin really able to detect sound??
-modifying silk worms?
-radiotrophic fungi (fungi that use gamma radiation to produce ATP - the pathway could be used in E. coli to absorb radioactivity but the exact pathway does not seem to be known (Wikipedia))
-serum bile acid as a biomarker of liver problems in pregnancy

Research for venom:
organise slides into problem, specifications needed to tackle problem, how to achieve specifications
-venom chosen: Asian cobra (one of the most common ones)
-Ming: summarise problem and specifications needed
-Chris: look at shark antibodies
-Nikki: look at in vivo mutagenesis (PCR mutagenesis would be too tedious)
-Frank: selection (FACS with GFP?)
-Rebekka: genetic circuit (detection mechanism - two component system?, suicide mechanism for cells that do not detect venom)

brain storming:

 

-        meeting with RCA people

 

-        presenting the DNA hard drive idea

1.         use DNA sequence as a medium to store information

DNA base - quaternary bit - original text

2.         put “tag in” information (i.e some specific base pairs as “prototypes”) to classify the information

3.         writing functions in programming language with DNA sequences, use DNA as the function variables

 

- Sci-Fi:

 a. in the future, a bacteria charm/necklace can be made to store and carry the information, such as exam syllabus, genetic disease history,  family photos, etc

 b. different coding methods and types of bacteria can be chosen by the clients for different levels of security needs ,(some high risky information can be store in Bacillus anthraci for military use)

 c. data can also be stored in E.coli in the human digestive system, it can be erased by intaking antibiotics

 

-        practical problem:

we need a bacteria platform to carry out the operation of the data

(I.e: bio-compiler? bio-CPU?)

a compiler consists of sets of programs and logic relations, it is theoretically workable, but obviously too difficult to do as a ten-week project

 

-  afternoon:

brain storming:

 

-        since the bio-compiler idea is abandoned, we thought about other ways to store data:

 

-        ROM memory

8x8 gird to store 64 bits of data

the bacteria interaction can be modified to give to outputs, which indicates the two binary states (0 or 1)

 

-        JK flip-flop or synchronized counter

 

-        7-segment display

one bacterium -four input channels-each channel with two states (0 or 1)-nine outputs to give nine numbers (0 to 9)- control the corresponding segments to display the number

-        bacterial minesweeper

an inhibitor can be considered as a ”mine”

the main problem is that secondary diffusion is very difficult to control. a mass transfer equation must be modeled for each individual square

 

Bile Acid Sensor:

-        the idea came up with SI’s final year project

-        a biosensor can be produced for daily home use to detect the bile acid concentration level in blood to prevent a series of liver diseases, especially the Intrahepatic cholestasis of pregnancy (ICP)

-        the basic concept behind this sensor:

bile acid - enzyme binding with the acid molecules – promoter – triggering the FXR gene – production of GFP

-        GFP is normally used as an indicator of the bio-sensor

the main problem is that the fluorescent intensity is very hard to quantified for a home-user

-  being inspired by the cosmetic skin colour sample card , we can make a sample card of fluorescence to give the rough concentration level of bile acid

-  a threshold value is required to tell the patient when their blood bile acid level is dangerous and may need a medical treatment

-  therefore, we will modify the linear relationship between bile acid concentration and GFP intensity level into a Hill system using Hill equation to find the threshold value

-   also, we may use colour indicator instead of GFP (light indicator)    

 

Christopher Schoene

Today we discussed the anti-venom idea with Koby (the RCA student that had arrived). He helped us expand the idea to maybe provide protection against viruses as well. We even developed the idea to use a seasonal hand wash containing the purified antibodies from the season's viruses in order to create a world where a handshake would be more than just a form of greeting but also a way to pass on immunity.

After the brain-storming session we had a talk from Chris about characterisation giving me a glimpse into what I would be expecting in the weeks after the project has been pinned down on a single idea. We currently have two running ideas; the anti-venom and a bile acid sensor. We have split off into two teams and each is working hard in order to build a system that the professors might agree to this Friday at 3:30pm. The deadline is approaching and we still require 2 more possible projects to present to the professors. I will work with my team to complete the anti-venom idea tomorrow and I will try to research a way to improve upon the idea of the use of yeast in water retention by making the express mucins.

RCA sci-fi story ideas:
Nuclear winter leaving people without immune systems. Use external immune system to save everybody?

Synthetic life can only use certain amino acids, firewall?
Biocomputers where man and machine are converged closer together.
Floppy disk baby?
Mock news articles (Times, science article, tabloid), comic strips (simplified version of our project), documentary type video?,
Terrorist attacks

Characterization presentation by Chris:

in vivo presentation
Timer vs. switch. need to know characteristics of parts.
How to characterise
Look at individual parts. What goes in, what goes out. What function is carried out.
What specifically do you want to characterise.
Terminator, core-promoter, RBS, reporter, coding region.
Terminator requires several different characterizations.
Place promoter, coding region into an expression circuit with a reporter.
Standards are only existant for promoters. How strong promoters are.
Canton paper 2008. Base characteristics that you need to know about an inducible promoter.
How responsive (transfer function of response), dynamic? (output)
All relative to J23101.
Synthesis rate, number of cells and divide your promoter output with standard which gives you a value relative to J23101. RPU (relative promoter unit)
RBS calculator gives you a value to how strong it is. Need to debug yourself.
Hard to characterise until we know what we want to do.
Plate readers look at OD, fluorescene, luminescence.
Individual cells analyzed for GFP and RFP.
Chris works on methods to make it more high-throughput (automates to make faster).
Won't consider optimizing something unless it worked twice on the assays.
Fluorescence doesn't give use pinpoint accuracy. Can't be measured really well, only bulk events and not specific events.
Enzymatic activity more done in vitro.
You're working with factories that work depending on what they're in.
Can find Chris though James at any time.
Make sure positive and negative control will work.

Antibody research:

-Single-domain antibodies such as the nurse shark derived IgNAR and the camelid derived VHH have been used for many purposes and have recently started to gain popularity among the scientific community.
-Contain CD3 loop that gives these Ig's an advantage when looking for cryptic viral epitopes. However, contain ten epitope copies and might still infect. Solved by increasing their mass.
-Both of these Ig's are stable enough to be administered orally.
-Orally administered transformed lactobacilli were used to administer anti-TNF Ig.
-Small dimensions of VHHs allow it to be easily tagged.
-Lactobacilli have been engineered that produces VHH's at a rate fast enough to prevent infection by p2 bacteriophage. Possibly use lactobacillus for screening and E. coli for secretion?
-VHH's and IgNAR's have been effective in detecting poliovirus and inhibiting its replication in vitro, as well as preventing the assembly and secretion of hepatitis B. Possible to use VHH's as intrabodies vs. HIV-1?
-N-glycosylation increases stability.
-Studies demonstrated that pre-immune libraries can be used for rapid generation of Ig's against a large number of harmful antigens. Troublesome low sensitivity overcome by using phage-displayed instead of purified antibodies.[1]

References:

[1]Ario de Marco, “Biotechnological applications of recombinant single-domain antibody fragments,” Microbial Cell Factories 10, no. 1 (2011): 44.

Frank Machin - A method is needed to sort cells by their ability to bind the venom proteins
- One such method would be to have the cells secrete their anti-venom proteins and then flood the cells with venom. The best cells would survive and the weak ones would be killed. However, this may cause the production of proteins that bind the venom components that are less dangerous such as phospholipases and oxidases. It would be more important for the anti-venom to inhibit the neuro-muscular disruptive proteins

- One effective method would be to use Fluorescence Activated Cell Sorting, which is able to sort cells by their fluorescence. See: http://www.bio.davidson.edu/courses/genomics/method/FACS.html
- This would require a system that expresses a fluorescent reporter in response to the binding of venom proteins to the cell-surface proteins, but I cannot be too specific until I know how the anti-venom proteins are going to be expressed
- Yet another method would cause the death of any cells that do not bind to the venom with high enough affinity - it would be similar to the methods employed in T-cell selection in eukaryotic immune systems, but would be more tricky as it has to occur in a bacterium
- The ideal then, would be to have all the cells that have little or no binding affinity killed, and then those that do bind express GFP so that the fluorescence can be quantified and the binding can be rated
- Again, I'd need to understand the anti-venom proteins before I could suggest any particular systems
- Perhaps a second species of bacteria could be used to express the venom proteins on their surface, and come into contact with the anti-venom producing cells, causing contact-dependant stimulation, so that those that interact by the venom-antivenom complexes will be stimulated to divide whilst the remaining cells will potentially be killed See: http://www.ncbi.nlm.nih.gov/pubmed/21085179

Nikki Kapp
- we need a method for random mutagenesis of peptides to create high affinity binding proteins to the multiple components of venom
- PCR based mutagenesis is namely used for site directed mutagenesis and has several biases that make it not ideal for random mutation
- in vivo homologous recombination inherent to yeast can be exploited for protein mutagenesis
(Pirakiticulr et al., 2010)
MAGE (multiplex automated genome engineering)
See: http://www.nature.com/nature/journal/v460/n7257/fig_tab/nature08187_F4.html - a method for large scale evolution of cells
- has been used to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in E.coli for isoprenoid lycopene overexpression with a pool of synthetic DNA to modify 24 genes in the pathway creating > 4.3 billion combinatorial genomic variants per day (Wang et al., 2009
- can do insertions, deletions and mismatches
See: http://www.nature.com/nature/journal/v460/n7257/fig_tab/nature08187_F1.html

Atipat Patharagulpong
How can venom antibody be engineered with bacterial platform using synthetic biology?

Venom refers to varieties of toxins produced by certain types of animals. One of the most common venoms are produced by snakes where 15% of 3,000 species of snakes are found poisonous. Snake venom consists of proteins, enzymes, substances with a cytotoxins, neurotoxins and coagulants

Most snake envenomings and fatalities are found in South Asia, South East Asia and sub-Saharan Africa with the high fatality rate of 125,000 deaths per annual. Among these India is reported the most cuased by big 4 including Russell's viper, Indian cobra, saw-scaled viper, and the common krait. Indian cobra is found the most famous and make the highest fatality rate (43%) in India and Southeast asia.

Indian cobra venom contains a potent post-synaptic neurotoxin which acts on the synaptic gaps of the nerves, thereby paralyzing muscles, and in severe bites leading to acute respiratory failure or cardiac arrest. The components of venom include lysis enzymes such as hyaluronidase which increase the spread of the venom. Its toxicity is found one of the highest based on LD50 value in mice. Symptoms of cobra envenomation can begin from 15 minutes to two hours after the bite, and can be fatal in less than an hour.

Despite the advance in emergency therapy, antivenom is often only the effective treatment. In treatment antivenom is injected into patient intravenously which could neutralize the toxin. Collecting of antivenom is done by milking the venom and injected into the cattle. The subject will undergo immune response where its antibody produced can be collected. This common method of obtaining antivenom can tackle many toxins in the venom but however is considered unproductive since only small amount of antivenom is produced from the animal blood which is due to complicated serum purification process from the animal's serum. Waiting of animal recovery from venom also make its production quite slow and in several cases the animals die after injection.

Therefore bacteria should be introduced as a substituted platform utilizing synthetic biology to deal with the problems mentioned above. Apart from existing characterised antivenom, other antivenoms could be more easily discovered using the various mutagenesis of variable region of antivenom antibody. To develop this platform, well known venom could be produced to test this platform. Indian cobra snake is therefore chosen as the first target due to its generality, high toxicity and it is also one of the highest profile antivenom discovered which could potentially save many victims from this fatal snakebite.

The way to engineer the bacteria is to mutagenise the shark antibody gene to allow different variable regions of the antibody to be expressed on the surface of the bacteria in different libraries. The venom is screened onto each plate of different bacteria libraries. The venom will bind to the right antibody and trigger the signal cascade which results in the expression of the fluorescence proteins which can be detected by FACs. machine. The bacteria that produces the antibody for the venom will be subjected to DNA sequencing which could be the platform for producing bacteria expressing antibody specific to the venom in the future.