Difference between revisions of "Team:UiOslo Norway/Description"
| Line 28: | Line 28: | ||
<p class="boxnotes leftext"> | <p class="boxnotes leftext"> | ||
| − | Today the second most common infection caused by bacteria is urinary tract infections, and the trend of this infections being caused by resistant bacteria has increased in the last years. | + | Today the second most common infection caused by bacteria is urinary tract infections, and the trend of this infections being caused by resistant bacteria has increased in the last years. ESBL-producing E. coli produce hydrolytic enzymes, β-lactamases, which have the ability to cleave and thereby inactivate antibiotics. |
</p> | </p> | ||
| Line 36: | Line 36: | ||
<p class="boxnotes leftext"> | <p class="boxnotes leftext"> | ||
| − | <b>Diagnostic test:</b> We will make a diagnostic test that will detect ESBL producing bacteria in urinary tract infections. The already established Penta Well test by Mura et. al. (2015), changes color from yellow to red when this resistant bacterium is present. The color change is due to the cleavage of the | + | <b>Diagnostic test:</b> We will make a diagnostic test that will detect ESBL producing bacteria in urinary tract infections. The already established Penta Well test by Mura et. al. (2015), changes color from yellow to red when this resistant bacterium is present. The color change is due to the cleavage of the β-lactamring in Nitrocefin. The test can also discriminate between several classes of β-lactamases, and we intend to base our idea on this. |
</p> | </p> | ||
Revision as of 16:19, 18 October 2016
Description
This year’s iGEM team from UiO will address one of the increasing world problems and health treat, antibiotic resistance. For many years humans have evolved together with bacteria in an evolutionary race, for each new antibiotic produced, a simple bacteria will eventually be able to resist the medicine, survive the treatment and continue to grow. Today we have come to a place where there is little or no new antibiotics produced and the resistance of the ones that are in use is increasing. Without functional antibiotics modern medicine will fall back many centuries. Without antibiotics doctors won’t be able to perform basic surgeries, keep diabetes patients healthy, and treat pre-term babies from infections. It is postulated that antibiotic resistance may become a greater threat to our health than cancer, and by 2050, a frightening number of 10 million people is estimated to die due to infections caused by antibiotic resistant bacteria.
Today the second most common infection caused by bacteria is urinary tract infections, and the trend of this infections being caused by resistant bacteria has increased in the last years. ESBL-producing E. coli produce hydrolytic enzymes, β-lactamases, which have the ability to cleave and thereby inactivate antibiotics.
Our goal is to make a diagnostic test associated with an app, which is available for doctors both in the field and in the medical offices. We’re in addition making a scheme of an screening test that fits the mention criteria.
Diagnostic test: We will make a diagnostic test that will detect ESBL producing bacteria in urinary tract infections. The already established Penta Well test by Mura et. al. (2015), changes color from yellow to red when this resistant bacterium is present. The color change is due to the cleavage of the β-lactamring in Nitrocefin. The test can also discriminate between several classes of β-lactamases, and we intend to base our idea on this.
Smartphone app: The diagnostic test will be associated with a smartphone app – phonelab. The camera on your smartphone will be connected to the app which by help of a binary code will detect any colorchange. Color will be cross-referenced with a database which will give the doctor a list of any resistant bacteria found and what antibiotics not to give.
Screening test: We want to use a simplified CRISPR/Cas9 system to link base-pairing to a detectable signal. The system will be comprised of a gRNA towards the dangerous resistance genes and a modified Cas9 protein. This will enable us to screen for the dangerous resistance genes, thus preventing antibiotics to end up in the wrong place even before the resistance gene products are already present. Conceptually; the gRNA is linked to a null-nuclease Cas9 that doesn´t cleave the target genes. Linked to the Cas9 is a detectable partner, potentially another protein. When gRNA binds to its target, Cas9 functions as a link between the gene and its partner. The bottleneck right now is to think of a detection method that will only detect the Cas9-construct when bound to the genes while being fast and easy enough to be relevant for our idea.
Urinary Tract Infection
This text will contain facts about drugs and treatments that have a base in Norway. Different countries often use different drugs and treatments.
Cystitis, urinary tract infection and painful bladder disease are all names for the same disease. About 80-90% of the time, the infection is caused by the bacteria Escherichia Coli (E. Coli) - a gram-negative bacteria that lives in our intestines. When E. Coli (or another bacteria) makes its way through the ureteral and to the urinary bladder, we have an urinary tract infection. Urinary tract infection can be divided into two categories; upper urinary tract infection (which include infection of the kidneys, also called pyelitis), and lower urinary tract infection (which only infect the ureteral and the bladder). You may also call it uncomplicated urinary tract infection or complicated urinary tract infection.
The infection affects more women than men. It is a 100 times more common for women to get cystitis, than it is for men. The reason for that is biology and anatomy. Women have a shorter ureteral and therefore it is easier for the bacteria to infect the bladder. Also, after menopause, women often experiences that the mucosa gets dry. This makes it easier for the bacteria to infect organs.
So, how do we treat cystitis?
In Norway we have national guidelines that doctors use in the treatment of different kinds of infection. The guidelines are made to keep the total amount of antibiotics down, and to give the best and most effective treatment to the patient. It is based on the principle that we must use narrow-spectrum antibiotics before broad-spectrum antibiotics. This is to keep the antibiotic resistance at a distance.
Uncomplicated urinary tract infections and complicated urinary tract infections are treated differently. The body can often take care of an uncomplicated cystitis by itself, but many use antibiotics to shorten the duration of the disease.
As many as 40% of the E. Coli found in urinary tract infections in Norway have been seen to be resistant against amino penicillin, such as amoxicillin. The other amino penicillin have shown to be intermediate sensitive, and therefore amino penicillin are no longer first hand treatment against cystitis.
Other penicillin such as mecillinam penicillin can still be used, as about 91% of E. Coli are still sensitive. Less than 2% are resistant.
Trimethoprim, along with pivmecillinam and nitrofurantoin are considered as first hand treatment against lower urinary tract infections. Usually there is no need to treat the patient for more than three days. According to the national guidelines for antibiotics in Norway the first hand treatment looks like this:
- Trimethoprim: 160 mg 2 times a day or 300 mg in the evening for 1-3 days
- Nitrofurantoin: 50 mg 3 times a day for 3 days
- Pivmecillinam: 200 mg 3 times a day for 3 days
All these treatments are considered as equally adequate treatment. Note that this guide is considered for adults who are not pregnant.
A new study also suggests that NSAIDs can be used in the treatment of lower urinary tract infection. It is considered to be equally as good as quinolones.
Treatment against pyelitis is the same as for uncomplicated cystitis, but the treatment is of longer duration.
- Trimethoprim: 160 mg 2 times a day or 300 mg in the evening for 5-7 days
- Nitrofurantoin: 50 mg 3 times a day for 5-7 days
- Pivmecillinam: 200 mg 3 times a day for 5-7 days
Note that treatment depends on severity and response of treatment. There are other guidelines for pregnant women and children.
The doctors also have to consider many factors when deciding which treatment is the most adequate for the patient; such as kidney function, allergies, pregnancy (which trimester?), breastfeeding, age and so on.
PhoneLab
APP description
The app developed for use with the PhoneLab was devised with the thought of measuring the color change of the urine samples by using the diode in our camera as the photoreceiver. Since the color change might be minute and only small amount of bacteria are antibiotic resistant we want to be able to register these small amounts of color change in the samples.
At first we considered a full spectrophotometer but decided that just taking a picture under the right lighting circumstances would be adequate to measure color changes quite accurately.
So the IT department designed the app based on these criteria and chose to develop the app with Android SDK using Java's API to handle the pixel readout and baseline comparisons that would have to be made to account for the different nuances of urine.
More precisely explained the app has to account for the base sample of urine and store the unique RGB value of the urine sample without any reagents, so that it can be compared to the samples with reagent. After this comparison is made the color change from the pixel’s RGB values are evaluated as either significant enough color change to determine antibiotic resistance or not significant enough color change to determine antibiotic resistance.
The reason for using a camera diode instead of just looking with the naked eye is because of the sensitivity of the camera combined with controlled light environment should equate to more reliable measurements.
APP design
The app was created in Android SDK and AIDE (Android IDE) it features very simplistic design with some information about antibiotic resistance to help doctors choose suitable antibiotic alternatives in the case of a sample containing antibiotic resistant bacteria towards conventional antibiotics.
The application takes a picture into the phoneLab device and runs a quick test on the resulting bitmap from the picture taken.
The points of reference are the 7 dots on the example picture above, where the bottom sample has no reagent added to the urine so that the bottom sample is the color change reference point. The color change is then measured from this baseline RGB value and compared with the 6 other samples to evaluate if there has been a significant color change in any of them.
Hardware
It all started with our iGEM team wanting to build a diagnostic device to deal with the problem of antibiotic misuse. After deciding we were going to work with beta lactamase enzymes in urinary tract infections, we started with our first idea; detection of changes in pH caused by hydrolysis of the beta lactam ring found in all beta-lactam antibiotics. To do this, we initially imagined to design and use an ISFET device (Ion Sensitive Field Effect Transistor), but due to poor readouts with our lab equipment we decided after a while to move away from the pH-measurements and go for an optical solution.
Figure 1. The first sketches of Prothenius - an ISFET device run by an arduino computer along with a simple display, to detect the hydrolysis of beta lactam ring.
The optimal candidate for optical detection of beta lactamase proved to be nitrocefin, a molecule with a beta-lactam ring that does not function as an antibiotic. When beta lactamase hydrolyse its beta-lactam ring, nitrocefin turns red. This enabled us to do an optical measurement of the color change to determine the presence of bacteria resistant to beta-lactam antibiotics.
The first idea to tackle this problem of measuring colorchange was to build a spectrometer using Arduino computer and 3D printing, but due to casts, this would have been less viable to use in for example third world countries.
We therefore decided to utilize the power that recides in the mobile devices many carry around every day; the smartphone. The modern smartphones generally have very good cameras, powerful processor calculations, and it is easy to develop apps for them. To do so, we needed to design and make hardware that allowed us to interface our samples with a mobile camera, and further design software (an app) to work with this hardware.
Figure 2. The first sketches of Phonelab. The left sketch is the very first version which was changed to the middle sketch due to some optics problems. The sketch to the right is a single cuvette test.
The purpose of this design is to create a stable way to interface samples with the app. The app collects all the information needed to read and analyse the color distribution between cuvettes, based on the contents of each tube. It then compares the results to a pre-made list containing information on what we are looking for. It would be convinient to have a login-option with an interface where you can add projects and patients, with the possiblilty to add notes. It could also be possible to allow doctors to send prescriptions for the suitable medicine. This could, for example, be done with a QR code on sms. For more information about the app, see the “Software”-section.
Figure 3. The first drafts of the app that would analyze the colors that phonelab detects.
As in any project, there were several prototypes before arriving at our final design. The assembly of the first prototype of PhoneLab was used to find design flaws and proving that the project was possible. The problems we encountered were the focusing on the cuvettes in such a way that we get the most accurate color readout, and the color difference of each diode. We decided that the next model would feature a different geometry to fix these issues.
Figure 4. The assembly of the electronics for PhoneLab prototype 1.
The dimensions and shape of the PhoneLab Optics design with 5 tubes for the nitrocefin penta well test are shown below.This design is made with an exchangeable phone adapter to fit with many different phones. A small LED is included in each slot, to ensure a homogenous and stable illumination of the samples. The LEDs run of an internally installed battery pack, and the light intensity is adjustable. As you see there is equal distance to every slot, and light from the LEDs are emitted radially through the slots where the sample is placed, towards the origin where the lence is placed.
Figure 5. The geometry of PhoneLab Optics, made for perfect 3D prints and cuvette exposure to camera.
To further improve on our device, we imagine to coat it with a hydrophobic layer that repels polar molecules, thereby ensuring it stays clean when being used. We also want this device to autoclavable without being destroyed. In this way it can be easily sterilized, when using it on bacterial samples, and it can be reused in a safe manner. The current material is PVA plastic, meaning that we would have to change the material to make it autoclavable. The cool part about this product, in addition to the box itself, is that it interfaces of biological samples to a phone and gives the userbase the ability to store, geotag, and share information in such an easy way through our software.
To summarize; through a series of prototypes, we have designed and 3D-printed a device that interfaces urine samples, or any sample one has software to and interest in analyzing, with our PhoneLab.
Design
Design here
Experiments
experiments here
Protocols
EQUIPMENT, MATERIALS AND SOLUTIONS
Pipettes
Pipet tips, assorted sizes
50 ml falcon tubes
Urine
MQ water
Spectrophotometer
Nitrocefin(41906-86- 9 Oxoid) and Nitrocefin reconstitution fluid (67-68- 5 Oxoid 5 Reconstitution fluid: Dimethyl sulphoxide (DMSO) 2mL)
Overnight E. Coli culture with amp resistance, (Bl21 plasmid)
Waste container for contaminated liquids
PROCEDURE
We always wear lab coat when working in the lab.
Overnight culture.
1. Label all containers to be inoculated, either plates or flasks/tubes with liquid medium. Flasks/tubes should be labelled by writing on autoclave tape to signify live bacteria and as a control of waste treatment to signify when the culture has been inactivated after use.
2. Transfer bacteria from culture to new media using applicable equipment, bacterial loops for bacteria in frozen sample or on plate, or sterile pipette for liquid culture.
3. Add also the appropriate antibiotic.
4. Close all containers to avoid unintentional exposure or contamination immediately after transfer of bacteria.
5. Mix or streak out depending on media type.
6. Incubate cultures at 37˚C in designated incubators for 12 – 18h.
7. Discard all contaminated loops and tips as bacterial waste and clean work area with 70% ethanol.
Preparing the Nitrocefin solution
1. Add 2mL of reconstitution fluid to Nitrocefin (powder) and shake well. Working concentration 500mg/ml.
2. Nitrocefin is light sensitive and should be kept in the dark.
3. Prepare the urine sample, dilute bacterial suspension with urine or other suitable buffers to obtain appropriate concentrations. Spectrophotometer measurements
4. Add suitable amount of the different dilutions of Nitrocefin to the urine and bacterial samples.
5. Transfer the sample into a kuvette with a pipette, often 1mL is enough.
6. Measure the absorbance in spectrophotometer at 486nm.
7. Wash the kuvettes for each new sample with dH2O.
8. Discard all contaminated consumables and liquids as risk waste and clean work area with 70% ethanol.
Transformation into chemically competent cells:
1. Thaw 100ul aliquot competent cells on ice.Flick gently once to mix. OBS! Do not vortex, as this can shear the cells.
2. Add 0.5-1 ul of plasmid DNA that is at 100 ng/ul concentration.
3. Flick gently to mix the solution.
4. Incubate on ice for 30 min.
5. Heat shock at 42C for 30s.
6. Incubate on ice for 2-3 min
7. Add 900ul SOC medium, grow for 1 hour at 37C This step can be omitted if plasmid encodes for ampicillin resistance.
8. Spin down gently (5 min at 3000g)
9. Remove 900 ul supernatant
10. Resuspend cells into 100 ul of media
11. Plate out onto plates with appropriate antibiotic for selection.
12. Incubate overnight at 37C and store at 4C afterwards.
This is a general restriction digest protocol that the Uioslo Norway team used frequently. Depending on how much DNA to digest, the amount of buffer and water will vary. If digesting 100ng DNA 0.5uL of restriction enzymes is recommended and the total volume should be 10 or 20uL. If digesting more DNA, increase also the total volume.
This protocol was used for the restriction enzymes XbaI, EcoRI, SpeI and PstI.
Restriction Enzyme: Usually 1uL
DNA: 100ng-1000ng
NEB buffer 2: 5uL – 10uL
BSA: 0.5uL
Nuclease free water: Up to 20uL – 50uL depending on how much DNA added.
Incubate the reaction on 37°C (check optimal conditions of enzymes used) for 1h. Heat kill the reaction on 80°C for 20min.
SAP treatment:
Shrimp alkaline phosphatase will dephosphorylate the 5’ end of DNA, and prevent the vector from religation.
We used SAP after every digestion reaction with vector.
1uL of SAP was added to each restriction reaction.
Incubate at 37°C for 30min and heat kill at 65°C for 5min.
Purification:
The restriction digest products should always be purified before proceeding to ligation.
Usually a spin column or gel extraction is performed.
UiOslo Norway team used Quiagen PCR clean up kit spin column, which worked nicely without having to lose too much DNA during the purification.
When using spin column, follow the protocol provided by the manufacturer.
Measure the DNA concentration before proceeding to ligation.
1. Prepare a “Phusion mix” with the total volume of 40 µl:
5X GC Buffer: 8 µl
10mM dNTPs: 0,8 µl
Phusion Polymerase: 0,5 µl
Forward primer: 2 µl
Reverse primer: 2 µl
Template 20ng/µl: 0,5 µl
H2O: 26,2 µl
2. Prepare the program on the PCR machine:
bilde herComment: Remember to adjust step 3 to match the specific primers used in the reaction. The annealing temperature depends on length and G-C content. If the annealing temperature is unknown, a gradient PCR should be performed.
3. When the PCR reaction is finished:
1. Add 1 µl DpnI into the “Phusion mix” and incubate at 50®C for 1 hour.
2. Do a PCR cleanup kit and follow the accompanied protocol.
3. Run the PCR product on a gel for verification.
EQUIPMENT, MATERIALS AND SOLUTIONS
Pipettes
Pipette tips
Quiagen miniprep kit
Eppendorf tubes
Centrifuge for microcentrifuge tubes
Vortex mixer
EQUIPMENT, MATERIALS AND SOLUTIONS
We always wear gloves and lab coats when working in the lab.
Plasmid isolation is carried out according to the protocol from the manufacture. The only deviation from the protocol is a reduction in the miniprep elution volume from 100 to 50 µl, to obtain a higher plasmid concentration.
Before the first use of the Miniprep kit, add RNase to the Buffer A1, and store at 4 °C. Add the indicated volume of ethanol to Buffer A4 and Buffer AQ.
In advance cultivate E.coli cells; P-SOP# Growing bacterial cultures.
Clean the table with 70% ethanol.
1. Cultivate and harvest bacterial cells. Pellet 1- 5 mL of bacterial cells.
2. Cell lysis.
3. Clarification of the lysate.
4. Bind DNA, add binding buffer.
5. Wash silica membrane.
6. Dry silica membrane, let the column stay on the bench for 1min.
7. Elute DNA, use smaller volumes to obtain higher concentrations of DNA.
100 mL lysis buffer with:
- 1% Triton X-100
- 0,1 M NaCl
- 10 mM Tris (pH 8,0)
- 1mM EDTA
How to make lysis buffer:
- Add 91 mL MQ H2O to a 200ml bottle
- Add 8 mL of Tris-HCl (pH 8) buffer (see recipe further down)
- Add 0,037g of EDTA
- Add 0,58g of NaCl
- Add 1 mL of Triton
How to make Tris-HCl(pH 8) buffer:
- Add 80 ml MQ H2O
- 1,2114 g Tris.
- Adjust the pH down to 8 by dropwise adding HCl (1M).
Numbers used in calculation:
- MW NaCl: 58,4428 g/mol
- MW Tris: 121,05 g/mol
- MW EDTA: 372,24 g/mol
Reference: Mura et al.
Proof Of Concept
Proof here
Demonstrate
Demonstrate here
Result
Results here
Experiment Notes
notes here
Contact
