Team:Lambert GA/Project

Project

OVERVIEW

The Lambert High School iGEM team from Suwanee, Georgia is a group of dedicated and self described nerds who love the challenges of doing science. We set out to further our work from our previous year’s project, as we never had the chance to express our protein, chitin deacetylase.

During the year 2013 we isolated the chitin deacetylase gene, CDA2 from Sacchromyces cerevesai. It has been shown that this gene could be expressed in E.coli successfully. CDA breaks down chitin, a naturally occurring polysaccharide into chitosan. Chitosan is widely used in a multitude of industries; including agriculture, aquaculture, health, biofilm production and water treatment. It is relatively expensive and current methods use strong acids and bases to deacetylate the chitin. Our idea was to engineer cells to accomplish this task. The cells could be grown and used in a bioreactor and the waste produced is acetic acid, which is another useful product. Current industries that produce chitosan are often located near coastal areas due to the natural abundance of shrimp shells and other exoskeletons. Unfortunately the wastewater form production can be polluting to the fragile ecosystems.

Chitosan has antifungal and antibacterial properties. We needed to engineer a system where we could turn on and off CDA production in such a way as to not kill our host cells. To this end we designed several constructs that would direct expression to the periplasm using a pelB tag combined with a Tet repressible promoter. We added GFP to the construct so that we might be able to visualize the proper expression in the periplasm.

After research and discussion with our mentoring team and Dr. Styczynski at Georgia Institute of Technology we chose a threefold approach to the design. Sometimes large engineered proteins have difficulty folding properly in the periplasm so we needed to address the issue using different placement of promoters.

The first design we call the bicistronic construct. We engineered the pelB tag and CDA under TetR promotion and the GFP under a lac inducible promoter. Having only the pelB tag and CDA in the periplasm would hopefully eliminate the difficulty with a 2000bp gene folding properly in the space. The GFP as a reporter would just let us know our transformations were successful and we could choose colonies for colony PCR using the color selection. The introduction of two independent genes however could have been a drain on the cell leading to early cell death.

The second design of the bidirectional is a way to use just a repressible promoter of TetR, but have the transcription of the two genes occur independently in opposite directions. There are several commercial plasmids that use bidirectional promoters to accomplish multi gene expression. This construct proved very problematic as we were ordering the gBlocks from IDT. Because the back to back promoter sequences were likely to form hairpin structures we had to order this in two parts using a multiple cloning site as a linker between the two TetR promoters.

The last construct of our project was a fusion protein consisting pf the pelB leader sequence, CDA and GFP all under the promotion of one TetR promoter. The benefit we aimed for with this sequence would be the ability to visualize the GFP in the periplasm of the cells. The large size though of the protein could lead to folding difficulties and then a complete loss of expression of either genes; CDA or GFP.

Our project if successful could lead to two significant advances, the first being a more environmentally friendly way to produce chitosan. Using engineered E.coli cells, we named Chitinite, to create to useful products of chitosan and acetic acid from waste shrimp shells would help save coastal ecosystems. A second significant contribution to science is our Biobricked bidirectional promoter system. This can help scientists with dual gene expression.

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Another highlight of our efforts was a Discovery Dialogue held in a partnership with the Atlanta Science Festival. We brought together on one stage, in an open forum; a legislator, ethicist, immunologist, FBI agent and scientist. The public was invited to ask questions and the concluding polls showed both an increase in the positive perception of synthetic biology, but also a request to hold another session this coming year.

MATERIALS & METHODS

Resuspension

  1. Centrifuge the tube for 3-5 sec in small centrifuge to ensure the material is in the bottom of the tube
  2. Add TE to reach a final concentration of 10 ng\ul
  3. Vortex briefly
  4. Put tube (use butterfly float) into water bath at 50° C for 20 minutes
  5. Briefly vortex and centrifuge

Double Digestion for DNA constructs and Plasmids (pSB1C3 and pSB1T3)

  1. Shake DNA gently to reach equal DNA concentration throughout the liquid.
  2. Add 33 uL of water to each PCR tube of DNA constructs; 38 uL of water to each PCR tube of plasmids
  3. Add 5 uL of 10x NEB buffer to each of the tubes
  4. Add DNA to each tube
    1. 10 uL Fusion
    2. 10 uL Bicistronic
    3. 10 uL Bidirectional 1
    4. 10 uL Bidirectional 2
    5. 5 uL pSB1C3
    6. 5 uL pSB1T3
  5. Add enzymes to each tube
    1. 1 uL of both EcoRI and PstI to Fusion tube
    2. 1 uL of both EcoRI and PstI to Bicistronic tube
    3. 1 uL of both EcoRI and Bam HI to Bidirectional 1 tube
    4. 1 uL of both PstI and Bam HI to Bidirectional 2 tube
    5. 1 uL of both EcoRI and PstI to pSB1C3 tube
    6. 1 uL of both EcoRI and PstI to pSB1T3 tube
  6. Incubate at 37°C for 5-15 min
  7. Stop reaction by heating up at 65°C for 20 minutes.

Ligation

  1. Ligate these DNA constructs with plasmid PSB1C3-2070
    Construct Vector(pSB1C3) DNA Buffer Ligase
    Bicistronic 4μL 15μL 19μL 1.9μL
    Bidirectional 1 4μL 4μL 11.5μL 1.15μL
    Bidirectional 2 4μL 4μL 11.5μL 1.15μL
    Fusion 4μL 13.5μL 17,5μL 1.75μL
    • Note: Bidirectional 1 and bidirectional 2 are both placed into the same tube so the vector, buffer, and ligase are only placed once into that tube. The bidirectional 1 and 2 have separate amounts that are placed into the tubes. (applies to both pSB1C3 and pSB1T3)
  2. Ligate these DNA constructs with plasmid PSB1C3-2070
    Construct Vector(pSB1C3) DNA Buffer Ligase
    Bicistronic 4μL 12μL 16μL 1.6μL
    Bidirectional 1 4μL 3.5μL 10.5μL 1.05μL
    Bidirectional 2 4μL 11.5μL 15.5μL 1.55μL
    Fusion 4μL 11.5μL 15,5μL 1.55μL

Nanodrop

  1. Use 1 uL of EB buffer to use as blanks. Measure the blank twice; second time to confirm.
  2. Measure the concentration of the DNA for each of the DNA tubes by placing 1 uL into the nanodrop.

Transform with competent E. coli Strains (NEB 10-beta competent or NEB 5-alpha competent)

  1. Thaw a tube of E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 uL of cells into a transformation tube on ice.
  2. Add 1-5 uL containing 1pg-100ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. DO NOT VORTEX!
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 5 minutes. Do not mix.
  6. Pippette 950 uL of room temperature SOC into the mixture.
  7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37°C
  9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
  10. Spread 50-100 uL of each dilution onto a selection plate and incubate overnight at 37°C.

Create Liquid Cultures

  1. Fill clean culture tube with 7 mL of LB
  2. Use clean inoculating lube to take a sample from colonies. (you want a few cells)
  3. Place inoculating lube into the culture tube and spin the lube, making sure to release some of the cells into the liquid
  4. Cover tubes with caps lightly/lightly parafilm
  5. Set in a shaking water bath for 37°C
  6. Grow for 18-24 hours

Mini-Prep

  1. Pellet 1-5 mL bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15°C-25°C)
  2. Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 uL Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for
  4. more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  5. Add 350 uL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless. Vortex 30 seconds.
  6. Centrifuge for 10 minutes at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  7. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 60 seconds and discard the flow through.
  8. Wash the QIAprep spin column by adding 750 uL Buffer PE. Centrifuge for 30-60 seconds and discard the flow-through. Transfer the QIAprep spin column to the collection tube.
  9. Centrifuge for 1 minute to remove residual wash buffer.
  10. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 30 uL Buffer EB to the center of the QIAprep spin column, let it stand for 5 minutes, and centrifuge for 1 min.
  11. Nanodrop to find out concentration of DNA.

Double Digestion

  1. Shake DNA gently to reach equal DNA concentration throughout the liquid.
  2. Add 36 uL of water to each PCR tube of DNA constructs
  3. Add 5 uL of 10x NEB buffer to each of the tubes
  4. Add DNA to each tube
    1. 7 uL Fusion w/ pSB1C3
    2. 7 uL Bicistronic w/ pSB1C3
    3. 7 uL Bidirectional w/ pSB1C3
    4. 7 uL Fusion w/ pSB1T3
    5. 7 uL Bicistronic w/ pSB1T3
    6. 7 uL Bidirectional w/ pSB1T3
  5. Add enzymes to each tube
    1. 1 uL of both EcoRI and PstI to Fusion w/ pSB1C3 tube
    2. 1 uL of both EcoRI and PstI to Bicistronic w/ pSB1C3 tube
    3. 1 uL of both EcoRI and PstI to Bidirectional w/ pSB1C3 tube
    4. 1 uL of both EcoRI and PstI to Fusion w/ pSB1T3 tube
    5. 1 uL of both EcoRI and PstI to Bicitronic w/ pSB1T3 tube
    6. 1 uL of both EcoRI and PstI to Bidirectional w/ pSB1T3 tube
  6. Incubate at 37°C for 5-15 min
  7. Stop reaction by heating up at 65°C for 20 minutes.

Gel Electrophoresis

  1. Loading buffer was added to each DNA sample.
  2. Gel box filled with TAE buffer until it covered the gel (1% agarose gel).
  3. Ladder was loaded into the wells of the gel.
  4. Samples are loaded into the wells of the gel.
  5. Gel is run at 90V until dye line is approximately 75-80% of the way down the gel. (Run for about 1-1.5 hours)
  6. Gel is placed in Fast Blast stain or other appropriate DNA stain (
  7. Use any device that has UV light and visualize your DNA fragments and confirm base pairs of DNA fragments.

RESULTS

puc19 positive control

bicistronic contract of pelB CDA with GFP in psb1c3

polycistronic construct pelB CDA with GFPin psb1c3

fusion construct of pelB CDA with GFPin psb1c3

negative control- no plasmid

MODELS

Determination of Chitosan in Liquid Samples using a UV-VIS Spectrophotometer

1.0 Purpose

This Standard Operating Procedure (SOP) describes a method for the determination of chitosan in liquid samples with a Ultravilot-Visible (UV-VIS) spectrophotometer.

2.0 Principle

Chitosan reacts with Reactive Red 4A dye to form a colloid which absorb light at 575 nm. The absorbance is relatively linear with the chitosan concentration in the range of 5 to 20 ppm.

3.0 Health and Safety

3.1 Wear goggles, a lab coat, and gloves at all times during the experiment.

3.2 Wear a dust mask when weighing out the solid dye powder to avoid inhalation.

4.0 Instrument Parameters and Labware

UV-VIS Spectrophotometer:
  • Slit width: 1 nm
  • Path length: 1 cm
  • Measuring wavelength: 575 nm
  • Cuvettes, glass or quartz (glass is for visible range only)

Weighing paper

Distilled water or deionized water

Class A glass flasks

Class A glass pipets

Transfer pipette

Eppendorf pipette and tips, 10-1,000 microliter range

10-15 ml centrifuge tube or glass tube

Watch/timer

5.0 Reagent and Standard

  • Chitosan: Low molecular weight, Aldrich or equivalent
  • Acetic acid 10%, Fisher Scientific or equivalent
  • Glycine-HCl buffer, pH 2.5: Fisher Scientific or equivalent (Keep refrigerated)
  • Reactive Red 4 A dye: MP Biomedical or equivalent

6.0 Reagent and Calibration Standard Preparation

  1. 1000 ppm Chitosan stock solution preparation
    1. Wash a 1-liter class A flask thoroughly. Rinse well with deionized or distilled water.
    2. Add 200 ml 10% acetic acid and approximately 500 ml water to the flask.
    3. Weigh 1 gram of chitosan using weighing paper and record the exact weight.
    4. Transfer the sample completely into the flask containing water and acetic acid
    5. Completely fill the flask to the mark with deionized/distilled water.
    6. Add one clean stir bar to the flask.
    7. Cover the mouth of the flask with parafilm or plug it with a stopper.
    8. After stirring for four hours, Invert the flask and stir for another 30 minutes.
    9. If there is any remaining precipitate at this point, filter the solution.
    10. The chitosan stock solution 1000 ppm is complete and ready for use.
  2. Chitosan Calibration Solution Preparation Using Volumetric Flasks
    1. 5 ppm Chitosan Standard

      Pipet 5 ml 1000 ppm stock solution into a 1-L flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    2. 7.5 ppm Chitosan Solution

      Pipet 15 ml 1000 ppm stock solution into a 2-L flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    3. 10 ppm Chitosan Solution

      Pipet 10 ml 1000 ppm stock solution into a 1-L flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    4. 12.5 ppm Chitosan Solution

      Pipet 25 ml 1000 ppm stock solution into a 2-L flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    5. 15 ppm Chitosan Solution

      Pipet 15 ml 1000 ppm stock solution into a 1-L flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    6. 20 ppm Chitosan Solution

      Pipet 10 ml 1000 ppm stock solution into a 500 ml flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    7. 25ppm Chitosan Solution

      Pipet 25 ml 1000 ppm stock solution into a 1-L flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

    8. 30 ppm Chitosan Solution

      Pipet 15 ml 1000 ppm stock solution into a 500 ml flask. Fill the flask to the mark with deionized/distilled water. Cover the mouth of the flask with a stopper or parafilm. Invert the flask to mix well.

  3. Chitosan Calibration Standard Solution Preparation using an Eppendorf Pipette

    If class A flasks are not available, Eppendorf pipettes can be used to prepare standards with a total of 4 ml for each concentration.

    In each 15 ml centrifuge tube, pipet the following various amounts of 1000 ppm chitosan stock solution and distilled/deionized water:

  4. Preparation of Reactive Dye Solution
    1. Weigh 0.075g of Reactive Red 4A dye powder using a weighing paper.
    2. Transfer completely to a 500 ml class A flask.
    3. Completely fill flask to the mark with water
    4. Add a stir bar to the flask. Cap the mouth of the flask with parafilm or a stopper and mix well. The solution is now ready for use.

7.0 Calibration Procedures

  1. Transfer 4 ml of each standard into a separate 15 ml centrifuge tube
  2. Add 1-ml of pH 2.5 glycine buffer to each tube
  3. Add 1ml of 75 ppm dye to each tube
  4. Cap the tubes and shake for 20 minutes.
  5. Prepare a blank sample using 4 ml of distilled/deionized water, 1 ml of dye and 1 ml of buffer. Shake for 20 minutes.
  6. Set the wavelength of spectrophotometer to 575 nm and warm up the spectrometer for at least 15 minutes.
  7. Fill a cuvette with the blank and wipe away any liquid on the outside surface of the cuvette. Do not touch the side of a cuvette in the light path.
  8. Put the blank in the spectrometer and zero it out.
  9. Fill a new cuvette with one of the standard solutions. Record the absorbance and repeat for all standards.
  10. Use Excel to plot the absorbances and create a calibration

8.0 Determination of Chitosan in a Liquid Sample

  1. Transfer 4 ml of the sample to a 15 ml centrifuge tube. Add 1 ml of 75 ppm red dye and 1 ml of glycine HCl buffer. Mix well, then shake the solution for 20 minutes.
  2. Zero out the spectrometer with a blank. Read and record the sample absorbance.
  3. If the sample absorbance is too high, dilute the sample until its absorbance fall within the linear range of the calibration curve.
  4. Read the concentration of the diluted sample from the calibration chart.
  5. If necessary, multiply the concentration by the dilution factor to obtain the chitosan concentration in the original sample.

9.0 Quality Control

  1. Run a 15 ppm chitosan standard for every ten samples, and at the end of a sample sequence.
  2. 10.2 Run a duplicate for every ten samples and at the end of a sample sequence.

10.0 Documentation

Record all data timely in a logbook or a notebook.

PARTS

Main purpose of all the constructs:

Chitin deacetylase, CDA was isolated from S.cerevisiae. It is normally found in the ascopore wall during sporolation. We hypothesized that if we added a pelB localization tag to the CDA the protein would fold correctly in the periplasm. All of the constructs contain these genes: The pelB leader sequence, mature CDA2 gene, and the eGFP gene. The pelB leader sequence is necessary because it lead the CDA gene into the periplasm. The expression of the CDA gene in the periplasm will be non toxic to the E. coli cells. The eGFP will allow us to physically see that the DNA constructs were successfully transcribed and translated. The CDA protein produced will then be available to use to break down chitin and convert it into chitosan which has many industrial applications.

How to purify and use:

The constructs were ordered online at IDT and they came as gBlocks. (each construct contained 1000 ng). The constructs were resuspended as the instructions that came with the gBlocks stated. Then, they were digested (to eliminate the biobricks) and ligated into pSB1C3 and pSB1T3 plasmids. Once they were ligated into the plasmids, they were transformed into competent E. coli cells so that the genes may be expressed.

Construct 1: Bicistronic

The bicistronic construct consists of two promoters: the TetR promoter and the P(Lac) IQ promoter. This construct creates two different mRNA transcripts. One transcript will consist of the pelB leader sequence and the mature CDA2 gene which are both expressed under the TetR promoter while the other transcript will have the eGFP gene which is expressed under the P(Lac)IQ promoter. The eGFP will remain in the cytoplasm while the pelB leader sequence will lead the mature CDA2 gene into the periplasm. This means that the entire E. coli will glow green if the DNA construct is transcribed and translated correctly.

Construct 2: Bidirectional

The bidirectional construct only uses one promoter: the TetR promoter. This serves as an advantage because unlike the bicistronic construct, only one antibiotic (tetracycline) is needed to control gene expression. The construct puts two TetR promoters back to back with a multiple cloning site in between them. One TetR promoter controls the pelB + mature CDA2 genes while the other controls the eGFP gene. Once again, this creates two separate mRNA transcripts and the eGFP will be expressed in the cytoplasm while the mature CDA2 gene is led into the periplasm by the pelB leader sequence, just like the bicistronic construct.

Construct 3: Fusion

The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.

NOTEBOOK

March

Atlanta Science Festival

Received foldscope from Stanford Prakash Lab

April

Organized Chick Fil A Biscuit sale fundraiser

Worked with Next Generation Focus to provide science education enrichment

May

Trained new members in basic lab procedures

June

Researched yellow stripe rust/ CRISPR cas9 project idea

Researched PelB tags for relocating chitin deacetylase to the periplasm

Emailed FBI agents for outreach project

Researched expression vectors

July

Narrowed down focus of research to the PelB tags

Designed constructs using 3a assembly method

Designed constructs using gene synthesis method

Decided to use gene synthesis constructs due to resource restraints

Met with Georgia Tech iGEM team; received assistance for construct design

August

Completed constructs

Added a fusion protein

Practiced transformation procedures

Practiced digest procedures; fixed errors in procedure

Fixed illegal restriction sites in plasmids

Researched bidirectional and bicistronic constructs

Designed and received constructs from IDT

Resuspended sequences from IDT

Received filters from Rosco

Designed team shirts and jackets

September

Completed wiki

Completed banner

Submitted parts to parts registry

Designed handouts and flyers for Jamboree

Designed "Lab Hacks" outreach project for high school labs

Completed presentation for Jamporee

Completed poster for Jamboree