Team:SUIS Alpha Shanghai/Experiment

Experiment

EXPERIMENT
Experiment Overview

After synthesis of our DNA from Genscript, we received DH5α, Glycerol Stocked bacteria containing two types of plasmids. One is in puc57, a high copy plasmid, containing our designed BBa_K2216000 part. Another one is in PCCI, a low copy plasmid, which was used during ligation of our synthesized complete composite part BBa_K2216003. This part was found to be unstable on high copy plasmid dur to its size. Our aim was to make 4 BioBricks, two basics parts, namely BBa_K2216001 & BBa_K2216002 as well as two composite parts, BBa_K2216000 & BBa_K2216003. Information for each part can be found below.

  • BBa_K2216000: A composite part with prefix + promoter + RBS + aldA gene + Terminator + suffix. The gene is coded on the puc57 plasmid, so we need it to be on the standard pSB1C3 plasmid.
  • BBa_K2216001: A basic part with aldA gene. Primers with prefix and suffix have been synthesized. We planned to use PCR to make the part from either the puc57 plasmid or PCCI plasmid sent to us.
  • BBa_K2216002: A basic part with Malonate-semialdehyde dehydrogenase. Primers with prefix and suffix have been synthesized. We planned to use PCR to make the part from PCCI plasmid sent to us.
  • BBa_K2216003: A composite part with prefix+ promoter + RBS+ aldA gene + RBS + Malonate-semialdehyde dehydrogenase gene + Terminator. It is coded on the PCCI plasmid, so we need it to be on the standard pSB1C3 plasmid.










Our Wet Lab Work  

Step 1.Growing single Bacterial Colonies:

 We wanted to culture bacteria on LB agar plates in order to obtain single bacterial colonies. First we grow the bacteria on agar plates containing the antibiotic ampicillin and pick out colonies that grew overnight to extract plasmid. Remember to label the plate not to confuse the two types of plasmids. Colonies containing the plasmid pUC57 (containing BBa_K2216000) was grown on agar containing the antibiotic amphcillin. Colonies containing the plasmid PCCI (contining BBa_K2216003) was grown on agar containing the antibiotic chloramphenicol. Plates were grown for 24 hours at 37 degrees Celsius. We then used our plate of single colonies to grow a large homogeneous population of our bacteria by growing in liquid LB broth. Approximately 1 single colony of bacteria was added to 10 mL of liquid broth. Tubes were grown overnight (12 hours) at 37 degrees Celsius with shaking.

Step 2.Plasmid Extraction: 

We used the Plasmid Miniprep Kit from Sangon Biotech LTD (Shanghai)and performed as per the manufacturer’s protocol, which can be found here:Miniprep protocol The aim of this step was to isolate and purify the plasmids from our bacterial colonies.

Step 3. Restriction Digestion

After purification of our plasmids from the cells, we cut them using two different restriction enzymes. The cut DNA would be later used for ligation to the shipping plamsid and for gel electrophoresis to check if our digestion was cut and accurate. Standard restriction digestion protocol was used: Restriction Digestion protocol. Double digestion using EcoRI and Pst1 restriction enzymes was performed using restriction buffer "O" from Sangon Biotechnology Ltd Shanghai. Important considerations: Thaw solutions and buffers if needed. If the liquids suspend at the side of the tubes, spin briefly in the microcentrifuge to collect all liquid at the bottom of the tube. Restriction enzymes should always be kept on the ice. Pipette in the following order:

  1. 7 μL sterile distilled water
  2. 10 μL plasmid DNA(assuming a DNA concentration of 50 ng/μL; We measured using a spectrophotometer. However, estimation can be approximated with the miniprep kit)
  3. 2 μL 10x restriction buffer (In this instruction we followed a total volume of 20 μL, the restriction buffer should be diluted 10 times of the total volume. To fit the condition of both restriction enzyme, we use the O buffer from Sangon as it showed 100% effiecency for both EcoRI and Pst1 restriction enzymes).
  4. 0.5 μL EcoRI, 0.5 μL Pstl 3. Mix by pipetting up and down. 4. Incubate at 37 °C in water bath for 30 minutes.
  5. Incubate at 80 °C for 20 minutes.
  6. For storage, store at - 20 °C freezer. Otherwise, proceed to gel electrophoresis.











Step 4. Gel Electrophoresis:

To confirm the success and accuracy of our restriction digestion, we performed gel electrophoresis analysis. Protocol is described below and the results of our analysis is found in Figure 1.

  1. -Prepare agarose gel:
  2. The concentration of agarose gel should be made according to the DNA length. We followed a 1:10 ratio. 1. Prepare 100 mL 1 x TAE buffer by adding 10 mL 10 x TAE buffer to a cylinder containing 90 mL distilled water. Mix well with a clean stir rod.
  3. Pour the 1 x TAE buffer into a flask.
  4. Add 1 g agarose powder into the 1 x TAE buffer in the flask.
  5. Place the flask inside the microwave and set at maximum heat for 1 minute. Pause the microwave every 10 - 15 seconds to give the flask a couple of swirls. This will help in dissolving the agarose and in preventing the solution from boiling over. Repeat as needed, until all agarose has dissolved.
  6. Wait for the agarose solution to cool down but do not wait too long as it may solidify inside the flask. - Assemble the gel cassette by sealing off the gel tray with tape. Place comb in the proper location.
  7. Add 4 - 5 μL DNA stain (e.g. Sybr Safe) to the cooled agarose. This enables us to view the gel under UV illumination. Mix by swirling.
  8. Pour the agarose solution carefully into the cassette. Avoid forming and trapping air bubbles.
  9. Wait for agarose to solidify.
  10. Prepare the DNA sample by mixing the following in a clean microcentrifuge tube to a total volume of 12 μL: 2 μL 6 x Loading Buffer, 4 μL DNA restriction digest, 6 μL distilled water
  11. Peel the tape off the gel and remove the comb carefully.
  12. Place the tray with the agarose gel in the tank for electrophoresis. Make sure the wells are located at the negative end of the electrical field. (DNA is negative and will move to the positive end)
  13. Fill the tank with 1 x TAE buffer. Cover the gel with a layer of buffer. -Load your DNA sample(s) and the DNA ladder into the wells. Make sure to keep a record of which sample went into which well.
  14. Hook up the gel tank to the power supply.
  15. Turn on the power and run the gel at 100 V for at least 1 hour.

Step 5. Extraction of DNA from gel

After the gel electrophoresis run, which confirmed a successful double digestion of our target on our plasmid, we need to cut the gel and retrieve our DNA parts. For this step we used the Gel Extraction Kit from Sangon Biotechnology Ltd (Shanghai) and performed the extraction using the manufacturer’s instructions found here:Extraction of DNA from Gel Protocol

Step 6. Ligation and Transformation to Competent Cells

Ligation of our parts onto the shipping plasmid (pSb1C3) was performed upon successful purification from the gel run. Ligation to Psb1C3 was performed by mixing ligation buffer, digested fragment of each part and the linearized shipping backbone provided to us in tyhe 2017 iGEM distribution kit. Standard iGEM ligation protocol was performed, for which a more detailed description of steps and volume/concentrations of solutions can be found here:Ligation Protocol


Transformation of our plasmid into DH5a component e.coli cells also followed standard protocol outlined by iGEM, of which more details can be found here:Transformation Protocol . The cells were grown on agar containing the antibiotic chloramphenicol. Successful transformation of pSB1C3 plasmid is indicated by growth of cells as the plasmid contains the gene for resistance to chloramphenicol.


The picture on the right shows bacteria containing the standard pSB1C3 plasmid successfully grown on the medium containing the antibiotic chloramphenicol, indicating to us that the plasmid has been successfully transformed into the competent cells DH5a.










Step 8. PCR of the Plasmid and Gel electrophoresis

 After successful ligantion and transforation of pSB1C3 into our cells, we needed to check tosee if the plasmid contains our designed parts. For this we were required to design and use forward and reverse primers to run a PCR of the plasmid. This step id to ensure that the plasmid transformed into the cell are not empty plasmids but that they do indeed, contains our parts.
Using the primers we designed (see below), we ran a PCR of the plasmid to confirm the plasmid transformed into the cell are not empty plasmid but contains our gene.
After plasmid extraction from the bacteria (using the same protocl as step 2), a PCR was carried out followed by another gel electrophoresis run to confirm the presence of our plasmid.
The figure on the left shows the resulting gel electrophoresis run. The lanes that are empty are the empty plasmids while the lanes that have bands are the plasmids that have our gene successfully ligated onto them. The results of this test confirms our procedure was a success and the plasmid pSB1C3 contains our designed parts.


Step 9. Plasmid extraction, Drying and Submission to the iGEM Plate!.

Once we had determined the positive test for the presence of our designed parts within our sample, we could extract the DNA from the gel (using the same protocols outlined in step 5), we could then dry the plasmid and prepare for shipping using the iGEM shipping plate provided in the 2017 distribution kit. We are thrilled that in our first year studying synthetic biology we have successfully built a biobrick (see parts section).










Our Team
Our team is composed of a group of young and motivated students with helpful advisors from Shanghai United International School Wanyuan Campus. We all love synthetic biology. We aim to make a contribution for the society and improve the food safety issue.
Our team is composed of a group of young and motivated students with helpful advisors from Shanghai United International School Wanyuan Campus. We all love synthetic biology. We aim to make a contribution for the society and improve the food safety issue.
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