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Frank Machin So the method for the anti-venom generation begins as follows:

1:

Take the venom proteins and bind biotin to either the C or N terminus so that it is able to be fixed to a large ferro-magnetic beads that are coated in streptavidin. Streptavidin has an extremely high affinity for biotin and the beads can be picked up with a neodynium magnet - so the bacteria with the 'antibody' on their surface that is able to bind to the venom proteins will be attached to the bead.
2:

The bacteria will have a plasmid that encodes a gene for a single-chain variable fragment, a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin. This will be displayed upon the surface of the bacterium and will be on a plasmid that has a very mutagenic effect as described by Rebekka.

Rebekka Bauer

novel in vivo mutagenesis mechanism:
-construct target genomic DNA with really strong promoter with retrovirus recognition sequence (R U5 PBS etc to ensure that only this is reverse transcribed)
-transcribe into RNA using a crap RNA pol that introduces mutations
-reverse transcribe into DNA using an overexpressed reverse transcriptase (overexpressed to ensure that this happens before DNA is degraded). The RT should be coupled to an inducible promoter to ensure that mutagenesis only takes place in bursts.
-insert into genome via homologous recombination using an equivalent of infusion enzyme (maybe RecA?)

Atipat Patharagulpong

Contacts of people from RCA and LSE

RCA
- Pei-Ying Lin : peiying.lin@networkrca.ac.uk
- Koby : yaacov.barhad@network.rca.ac.uk
- CJ : charlotte@artforeating.com

LSE
- Alex Hamilton : r.a.hamilton@LSE.ac.uk
- Claire MArris : c.marris@lse.ac.uk
- Stephan Guttinges : s.m.guettinges@lse.ac.uk

Talk from LSE
Things to consider in human practise
Safety, Security, IP-intellectual property, ethical, biohacking, global fairness
For Anti-venom project
Safety : handling toxin in the lab, storage and transportation of toxins
Security : produce specificity problem, antivenom target for particular person
Patent : only the process obtaining product(antibody) but not the product itself, patent cost a lot of money

Idea : bacteria that solidify the soil in the presence of urea
Sporosarcina pasteurii or Bacillus pasteurii from older taxonomies is a bacteria with the ability to solidify sand given a calcium and an organic nitrogen source through the process of biological cementation. This will be a good recycle of land waste, urea waste and a food waste.
However solidification requires a high pH and produce toxic ammonium waste. Even though ammonia increase the pH this should be control using synthetic biology to model the right amount. ammonium can be subjected to other products which we are still searching for. Another application might be using ammonium produce to tighten the dye we made using the pigment which might allow the full house to be made easily from the brick.
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Si, Nina, Nick and Yuanwei

MORNING:-

Serum Bile Acid Sensor Presentation:-

1. Background:

- Bile acids are 24-carbon steroids found in bile, which are subject to enterohepatic circulation

- synthesized in liver and stored in gallbladder

helping in digestion and absorption of dietary fat and liposoluble vitamins

- bile acids = highly toxic

therefore their concentration must be tightly regulated

- the level of bile acids is controlled through a negative feedback system

mediated by a nuclear bile acid receptor FXR.

FXR is highly expressed in liver, intestine and kidney cells

it responds to bile acids and has been shown to repress CYP7A1, a key gene associated with bile acid synthesis.

- Defects in bile acid homeostasis due to functional variations of FXR

result in cholestatic conditions such as Intrahepatic cholestasis of pregnancy (ICP)

ICP is a pregnancy-specific liver disorder

characterized by pruritus (intense itch) an abnormal liver function

- The dysfunction of maternal liver could induce stress on the fetal liver as the fetus relies on maternal liver to remove bile. It has been shown that ICP pregnancies are more likely to suffer from meconium staining of the amniotic fluid (MSAF), cardiotocography (GCT) abnormalities and respiration distress syndrome (RDS).

- The risk of complications such preterm labor, prenatal death and stillbirth are directly linked to severity of ICP

- The morbidity of ICP is geographically and racially dependent. [1]

Country	Morbidity(%)
Sweden	4.2
Finland	1.0
Poland	1.5
Jugoslavia	1.1
Spain	1.6
UK	1.0
China	4.4
Aymara	13.8
Araucanian	27.6
Chile	15.6
Caucasian species	9.8

2. Mechanism:-

FXR based activation:-

- use eukaryotic transcription factors in bacterial gene expression

- suggested use of FXR (known structure and DNA sequence) with constitutive promoter expression

- a receptor protein of a number of bile acids, which would bind to ligand-bile acid

- can act as a repressor or an activator, we would use it as an activator, binding to a promoter region B4-BARE (2.4kb) taken from a gene UGT2B4, (known primers for promoter region)

- specified exact FXR binding site, therefore possibility of integrating FXR binding site into another regulatory region

- upon binding to bile acid it would activate output gene

- In the experiment using FXR as transcription factor in human cells 30mM DCA was used, however presumably lower concentration should be sufficient for triggering of FXR.

3. Output:-

- GFP:-

fluorescence = light indicator

= hard to quantify

- ligaments:-

colour = clear indicator

= influenced by the red colour of blood cells

= cell free mechanism for hospital use = accurate measurement

= filter/ membrane mechanism for home use

- protein expressed on surface of bacteria causing aggregation

(either of bacteria or of bacteria to a substance in blood)

4. Modelling:-

- Binding of activator to GFP gene is a positive cooperative reaction.

- Once activator molecule is bound to the enzyme, its affinity for other activator molecules increases.

- Hence, Hill equation can be used as a model.

Hill function for transcriptional activation:

k₁: Maximal transcription rate

K_m: Activation coefficient

n: Hill coefficient

A: [activator]

- This equation gives the % bound by activator as a function of activator concentration.

- after Hill equation modification, the system behaves like a switch

- In addition, we also need to model the diffusion of blood and GFP in the bacteria culture

5. Cell –free mechanism:-

- for hospital use, the blood must be processed through a cell-free system to give an accurate test result, as well as the bacteria to reduce the risk if they leak from the container ( after the cell-free process, the bacteria are not able to reproduce)

- separate certain organelles from whole cells for further analysis of specific parts of cells

- in the process, a tissue sample is first homogenised to break the cell membranes and mix up the cell contents

- homogenization is intensive blending of mutually related substances or groups of mutually related substances to form a constant of different insoluble phases

- homogenate is then subjected to repeated centrifugations

- each time removing the pellet and increasing the centrifugal force

- Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces.in the separating order in actual application:

Whole cells and nuclei;

Mitochondria, lysosomes and peroxisomes;

Microsomes (vesicles of disrupted endoplasmic reticulum); ribosomes and cytosol.

6. Filter/semi-permeable membrane for the blood cells:-

RBC: 7~8.5μm

neutrophil: 10~12μm

eosinophil: 10~15μm

basophil: 10~12μm

monocyte: 14~20μm

lymphocyte: small: 6~8μm, medium9~12μm, large: 13~20μm

taurocholic acid molecule roughly 0.4nm

- therefore, if the hole diameter of the filter is set to be at about 2~5nm, all the blood cells can be filtered

AFTERNOON:-

LSE BIOS sessions about human practice:-

A. why biosensors ?

- possible to construct

- has a potential market

-

B. human practice

1. bio-safety

- cell-free system to stop the bacteria from reproducing if there is a leakage of the sensor device

- (unpredictable) mutation must be carefully prevented during the engineering part

2. bio-security

- the access to DNA sequences and other genetic information must be controlled

- “garage biologist”

- the restriction to synthetic biology knowledge is not the way to prevent bio-terrorism, the key thing is the professional and correct guidance

3. IP (intellectual property issue) and patent

4. ethical and philosophy

5. global fairness

- bio-sensing = faster disease detection

- employment problem? discrimination?

- classify the diseases into different levels of risks

- social coordinate organization to optimize the occupation and personnel resources

C. LEGO exercise:-

- different ways to describe the construction process

- engineering language and user-friendly language

new idea

inflammation detecting bandage:-

- inflammation is caused by the immune response to pathogens

- can be acute to chronic

- acute inflammation:-

1. increased movement of plasma and leukocytes (granulocytes) from blood to injured tissues

2. causative agent = pathogens and injured tissues

3. cells involved:

neutrophils, mononuclear cells (monocytes and macrophagens)

4. primary mediator = vasoactive amine and eicosanoids

- to carry out the detection mechanism, we will set a target chemical to detect

- at this stage, interferon gamma is taken into our consideration

Interferon-gamma (IFN-γ) is a dimerized soluble cytokine that is the only member of the type II class of interferon.

This interferon was originally called macrophage-activating factor, a term now used to describe a larger family of proteins to which IFN-γ belongs. In humans, the IFN-γ protein is encoded by the IFNG gene.[2]

- the goal of this project is to find a gene coding the protein which and react with interferon gamma and give an indication

- a further step may be taken as a “damaged tissue cleaner”, which means that the bacteria can not only detect but also remove the inflammation tissue

[1] http://baike.baidu.com/view/676699.htm

[2] http://en.wikipedia.org/wiki/Interferon-gamma

Christopher Schoene

More Ig research:

To generate cell surface anchored antibody fragments: VHH sequence is fused to the anchor sequence from proteinase P of L. casei.

To generate the secreted VHH, stop codon after E-tag.

Sand brick research:

Sporosarcina pasteurii --> urease

Calcium carbonate bricks can be made by using urease, urea, sand and calcium chloride. The urease cleaves the urea and creates ammonia and carbon dioxide. The ammonia raises the pH and causes the calcium ions to precipitate with the dissolved carbonate ions.

Ammonia is currently a problem since it can run off to cause algal bloom. A method to remove ammonia must be obtained. Solution? Possibly express all the urea cycle enzymes in the chassis (5 total enzymes). Use slovenia's DNA scaffold so that the process does not produce too many possibly problematic intermediaries in the bacteria.

When should ammonia be converted back to urea? When calcium is depleted. Need some sort of Calcium sensor.

New way of transforming bacteria idea:

New way of selecting for transformed bacteria.

Create a plasmid containing Dpn1 under the influence of a strong repressor. In the same plasmid insert the methylase enzyme. The gene is inserted into the methylase and stops its transcription.

If the gene of interest has been successfully inserted into the plasmid and the plasmid has been successfully inserted into the bacteria we can use the inducer to kill off any bacteria that has not been transformed (because the Dpn1 cuts any methylated strands of DNA).

Today we continued developing the bile acid sensor as well as the idea of the anti-venom. For the anti-venom idea we talked to Travis Bayer and obtained a method for screening for the right mutants. We also had a visit from LSE BIOS members that taught us about the point points we should worry about in human practices. We were also given the task to make instructions for a lego structure to show us how difficult it is to standardize parts due to the difficulty of communicating more complicated instructions.

@@ Line 293: / Line 293: @@
 <DIV style="MARGIN: 0cm 0cm 0pt" class=MsoNormal><SPAN lang=EN-US><FONT color=#000000><FONT size=3><FONT face=Calibri><SPAN style="mso-spacerun: yes">&nbsp;</SPAN>[1] </FONT></FONT></FONT><A href="http://baike.baidu.com/view/676699.htm"><FONT color=#800080 size=3 face=Calibri><A href="http://baike.baidu.com/view/676699.htm">http://baike.baidu.com/view/676699.htm</FONT></A></SPAN><SPAN lang=EN-US><FONT color=#000000 size=3 face=Calibri></FONT></SPAN></A></DIV>
 <DIV style="MARGIN: 0cm 0cm 0pt" class=MsoNormal><SPAN lang=EN-US><FONT color=#000000 size=3 face=Calibri><FONT color=#444444 size=2 face=Arial>&nbsp;</FONT>[2] http://en.wikipedia.org/wiki/Interferon-gamma</FONT></SPAN></DIV>
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+<html>
+<b><br><br>Christopher Schoene<br><br></b>
+More Ig research: <br><br>
+To generate cell surface anchored antibody fragments: VHH sequence is fused to the anchor sequence from proteinase P of L. casei. <br><br>
+To generate the secreted VHH, stop codon after E-tag. <br><br>
+<b>Sand brick research:</b><br><br>
+Sporosarcina pasteurii --> urease<br><br>
+Calcium carbonate bricks can be made by using urease, urea, sand and calcium chloride. The urease cleaves the urea and creates ammonia and carbon dioxide. The ammonia raises the pH and causes the calcium ions to precipitate with the dissolved carbonate ions. <br><br>
+Ammonia is currently a problem since it can run off to cause algal bloom. A method to remove ammonia must be obtained. Solution? Possibly express all the urea cycle enzymes in the chassis (5 total enzymes). Use slovenia's DNA scaffold so that the process does not produce too many possibly problematic intermediaries in the bacteria. <br><br>
+When should ammonia be converted back to urea? When calcium is depleted. Need some sort of Calcium sensor. <br><br>
+<b>New way of transforming bacteria idea:</b><br><br>
+New way of selecting for transformed bacteria. <br><br>
+Create a plasmid containing Dpn1 under the influence of a strong repressor. In the same plasmid insert the methylase enzyme. The gene is inserted into the methylase and stops its transcription. <br><br>
+If the gene of interest has been successfully inserted into the plasmid and the plasmid has been successfully inserted into the bacteria we can use the inducer to kill off any bacteria that has not been transformed (because the Dpn1 cuts any methylated strands of DNA). <br><br>
+Today we continued developing the bile acid sensor as well as the idea of the anti-venom. For the anti-venom idea we talked to Travis Bayer and obtained a method for screening for the right mutants. We also had a visit from LSE BIOS members that taught us about the point points we should worry about in human practices. We were also given the task to make instructions for a lego structure to show us how difficult it is to standardize parts due to the difficulty of communicating more complicated instructions.
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