Team:Carnegie Mellon/Notebook

Under Construction.

The CMU iGEM team did a lot this summer from building a light to playing with an Arduino to improving an estrogen sensor. Luckily, we tracked everything in our handy-dandy notebook.

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Week 4

6.12.15

6.15.15

6.16.15

6.17.15

6.18.15

6.19.15

Week 5

6.23.15

6.24.15

6.25.15

6.26.15

Week 6

6.23.15

6.24.15

6.25.15

6.26.15

Week 7

7.6.15

7.10.15

Week 8

7.13.15

7.16.15

7.17.15

Week 9

7.20.15

7.21.15

Week 10

7.27.15

7.28.15

7.29.15

7.30.15

Take me to Tab 2 Take me to Tab 3

May 26th, 2015
  • First day of work!
  • Familiarized ourselves with the lab.
  • Made LB (protocol) & poured plates (protocol).

The light fluoresces! We have transformants & isolated proteins.

Week 3

6.8.15
Goal: To begin quantifying fluorescent proteins produced in MACH cells: mRFP, OFP, YFP, eGFP, BFP. Also to create a strongly expressing culture of YFP
  • Created 5 mL cultures from previous cultures, with the exception of the YFP transformed bacteria, which was taken from a plate created 5/27/2015.
  • Streaked a new multicolor plate with all his tagged fluorescent proteins
  • Calculated OD of 1:10 dilution of each culture using online calculator
      OD was 0.369, thus the non dilute concentration of bacteria we used was 2.95 * 10^9, and the amount of bacteria in each sample was 4.425*10^9
  • His-tagged soluble protein extraction outlined in our procedure using a washing solution with dilute imidazole
      Used 50 uL of beads for each
      extracted proteins from 1.5 mL of each culture
  • OD was 0.369, thus the non dilute concentration of bacteria we used was 2.95 * 10^9, and the amount of bacteria in each sample was 4.425*10^9
  • Read fluorescence in a microplate reader insert results
  • YFP is still not expressing (pictured below), and we did indeed create a culture from a weak colony insert picture
For next time: We will test our new cultures, which should be expressing well, as we marked strong colonies on the initial YFP plate. We will try a pH elution after extraction on Ni beads, as adding too much imidazole will interfere with fluorescence readings

Week 1

5.28.15
Goal: To do Miniprep to purify the DNA. It is a small scale isolation of plasmid DNA from bacteria. Then do restriction enzyme digest on plasmid DNA and gel electrophoresis. This technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of enzymes isolated from bacteria called restriction enzymes. They are able to cleave DNA molecules at positions at which particular short sequences of bases are present.
  • Did MiniPreps of MACH cells expressing fluorescence to isolate plasmid DNA from MACH cells (protocol in the Protocol Page )
  • Performed restriction enzyme digestion on J23108, J23109, J23111 constructs
      For promoters, PST1 & Spe1 were used. For insert, xba1 and Pst1 were used.
  • Ran an Agarose Gel Electrophoresis
      Expected size of insert (from E0040) - 720 bp
      Expected size of vector backbone (J23108, J23109, J23111) - 2105 bp
  • Cut digests from gels (protocol in the Protocol Page ).
For next time: Will prepare Colony PCR to determine if we got the correct restriction sites and sequences from the given interlab devices.

Week 2

6.01.15

Goal: To submit minipreps for sequencing, will get results in two days and compare our constructions.
  • Did minipreps on samples where the devices were incorporated in (J promoters & I3504)
  • Sent minipreps off for sequencing.
      5 µL water, 5 µL sample, 1 µL of each primer

6.02.15

Goal: Make competent MACH cells.
  • Made competent MACH cells (protocol in the Protocol Page )
  • measured fluorescence of GFP for 6 samples using a TECAN SaphireII Microplate Reader
      108A, 108C, 109A, 109C, 111A, 111B
    Parameter Description
    Microplate Reader Model TECAN Sapphire II
    Software XFlour4
    Measurement Mode Flourescence Bottom
    Excitation Wavelength 500 nm
    Emission Wavelength 515 nm
    Excitation Bandwidth 5 nm
    Emission Bandwidth 5 nm
    Gain (Manual) 150
    Number of Reads 100
    Flashmode High Sensisitivity
    Integration Time 40 µs

    Sample 108A 108C 109A 109C 111A 111B
    Reading 5932 6139 6050 5789 5589 6109

For next time: Since we did it without MACH cells, will redo tomorrow with MACH cells and biosensor to compare results.

6.03.15

Goal: Observe Sequence Data and compare to our own plasmids. Redo TECAN results with blank and biosensor to see differences in fluorescent data.
  • Compared sequence data to registry/our own plasmids. Figured out that no promoters got inserted in. Decided to send in Minipreps of the parts of the original devices for sequencing to see if the given parts were what we expected.
  • Redid fluorescence reading
      Very evident difference, shows the presence of GFP
      May redo in the future in order to do the trianalysis.
      Used same TECAN parameters as 6.02.15
    Sample Control 108A 108C 109A 109C 111A 111B Last Year
    Reading 3379 6914 6097 6381 7696 5949 6814 OVER

6.04.15

Goal: Streaked out plates, continued competent cell protocol.
  • Streaked out MACH cells, Top10, and Biosensor Estrogen - on to LB plates. The biosensor estrogen negative plates had kanamyacin antibiotic on it (10 ul + 50 ul water).
  • Began TOP10 Competent cell protocol (protocol in the Protocol Page ).
      Diluted TOP10 overnight culture 1:100 for a 2ml volume in LB. Grew it from 10a-5p. Then added 300 ul to each 500 ml of LB in sterile 2L flasks. Placed in 18C shaker overnight after removing the magnetic stir rod.

6.05.15

Goal: Get back plasmid sequences to determine what went wrong with what we did. Restreaked the plates and competent cells growth is slow therefore will continue procedure Monday 6/8/15.
  • Placed growing TOP10 cells in 2 L flasks in cold room because it was taking too long to grow in 18C shaker. Will place back in shaker Sunday night so that it can be ready on Monday to use (need OD600 of 0.4-0.6.)
  • Further sequencing results on original devices sent out on 6/3/15 were obtained on 6/5/15. It showed that the promoters have 2 additional Spe1 cut sites which meant that they were cut out, which is why none of the transformants had the promoter in them.
      We notified iGEM, and they will thank us on their website for finding their mistake.
  • Started Transformations (protocol in the Protocol Page ) for New Set Today.

Week 3

6.07.15

Goal: Set up culture preps for minipreps
  • Set up culture preps for minipreps (5ml) with 5ul of chlorophenical.
      Samples: 106A, 101A, 101B, 101C, 101D, 117A, 117B, 117C, 117D
      Left overnight in 37C shaker.

6.08.15

Goal: Redo the interlab study with the new devices given.
  • Forgot to do ligation and transformation, therefore we will redo this tomorrow.

6.09.15

Goal: Redo digestion of the new transformations and determine if they fluoresce.
  • Redid the Digestion because we forgot to do transformations (protocol in the Protocol Page ).
  • Did 3504 digestion with 101B, 106A, 117C
      For promoter: 10 µl of MiniPrep, 4 µl of H20, 2 µl of green buffer, 2 µl of EACH enzyme (PST and SPE1)
      For insert: 12 µl of MiniPrep, 2 µl of H20, 2 µl of green buffer, 2 µl of EACH enzyme (PST and Xba1)
  • Put digests into a gel & washed gel with 1:1 buffer and put it into 42C heat shock for ~30 minutes
  • Did PCR purification
  • Ligation done using 4 µl of promoter, 4 µl of insert, 1 µl of buffer (t4 ligase buffer) LIG, 1 µl of T4 ligase (protocol in the Protocol Page ).
  • Did transformation for lacYFP, FFluc, PRE, POST (protocol in the Protocol Page ).
      For lacYFP (can plate): 35 µl of cells, 1 µl YFP
      For FFLuc, PRE, POST (5mL LB AMP cultures): 35 µl of competent cells, 1 µl of such samples
      For our samples: 50 µl of competent cells, 10 µl of our mix (3504-101B, 106A, 117C)
  • Also redid competent cells (autoclaved the 500mL LB, store TOP10 into 37C put in around 1microliter ← slow growth so put in more in order to grow faster)
For next time: See if our devices are green fluorescing tomorrow and to fixate our cells to the W strain in the competent cells being made.

6.10.15

Goal: Screen the transformants for ones that uptook the plasmid and to check if competent cell culture is ready.
  • Screened the plates with transformants using UV light.
      Screened the plates with transformants using UV light.
  • Set up 5ml culture using one colony from each (106A, 101B, 117C) for miniprep. Grew overnight in 37C shaker. Picked colony based on fluorescence using the UV light.
  • Competent cells still did not reach the appropriate OD (currently 0.1 at 9:30a). Allowing it to shake overnight again.
For next time: Purify and isolate the transformant DNA using miniprep on the cultures that were set up today.

6.11.15

Goal: Purify and isolate the transformant DNA using miniprep on the cultures and then send it out for sequencing. Begin the competent cell protocol.
  • Did miniprep (protocol in the Protocol Page ) on the 5ml cultures containing colonies of 101B (J23101 + I31504), 106A (J23106 + I31504), and 117C (J23117 + I31504)
  • Sent out the DNA for sequencing.
  • Competent cell culture was still did not have an OD of 0.4-0.6. Will check again tomorrow morning.
For next time: Begin PCR amplification on Gaussia Luciferase using Phusion high fidelity DNA polymerase.

6.12.15

Goal: Finish competent cell protocol and start the Gaussia Luciferase experimentation.
  • Competent cell culture was within the OD. One of the competent cell cultures were within the appropriate range (at OD of 0.48). The other one wasn’t (at OD of ~0.2). We just started the competent cell protocol on both cultures but kept them separate so that we could compare their efficiency. The “Making competent MACH T1 cells” protocol was used for making competent TOP10 cells (protocol in the Protocol Page ).

Week 4

6.15.15

Goal: Verifying the sequences obtained to the constructs that were made. Check the competence of our competent cells.
  • Verified by comparing the sequence of our constructs to the sequence got from the samples we sent out.
      They seemed to be accurate with a few changes.
  • Checked competence of competent cells using the iGEM competent transformation efficiency kit.
      Tubes labeled 1 w/ X conc are from the culture with OD within the range of 0.4-0.6. Tubes labeled 2 w/ conc are from culture that did not have an OD within the range of 0.4-0.6.
      Tube 1 with concentration 0.5, 5, 10, 20, and 50 picogram (5 samples in total)
      Tube 2 with concentrations 0.5, 5, 10, 20, and 50 picogram (5 samples in total)
      Tube with MACH cells.
For next time: Will count the number of colonies and determine the transformation efficiency of the cells.

6.16.15

Goal: Verifying the sequences obtained to the constructs that were made. Check the competence of our competent cells.
  • Found nothing in both our competent cell plates and MACH plates from yesterday, using the DNA iGEM transformation efficiency kit.
  • Redid Competent cells and MACH cells with plasmid DNA
  • Plated both 400 µl and 40 µl for each competent cell transformation (total: 6 plates) & put in 37 °C incubator.
For next time: Check if our competent cells worked.

6.17.15

Goal: Verifying if our competent cells worked since it didn’t work using the DNA from iGEM.
  • Checked the plates and found cells in both MACH and competent cells.

6.18.15

Goal: Transform colonies into TOP10 Competent Cells.
  • Transformed colonies (101B, 106A, 117A) into TOP10 Competent Cells and then plated them.
      1 µL of DNA, 50 µL of cells, 5 min ice, 2 min heat, 5 min ice, 500 µL LB, 1 hr at 37 °C, plate

Week 5

6.23.15

Goal: Make cultures and plate cells.
  • Made 2 mL overnight cultures of cells that will later be used in the microplate reader to measure fluorescence.
      MACH empty, Top 10 empty, 3 devices + top 10, 3 devices + mach
  • Plated both the mach and competent cells overnight on LB plates to see if they would grow to use for the biological and technical replicate part of the interlab.
      Technical is more how many times we repeated something.
      Biological is doing new sets of colonies.

6.24.15

Goal: Measure fluorescence for MACH and competent cells.
  • Took out Mach and Top10 Competent Cells to do measurements of flouresence with on a TECAN microplate reader.
  • Used same TECAN parameters as 6.02.15 except used a Gain of 100 instead of 150.

Estrogen Sensor:

  • tried newly transformed cells, all taken from the same plate
  • tried to get similar-sized colonies
  • tested the following amounts of estrogen:
    • 100 uM, 20 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 0 nM
  • got very varied and inconsistent results
  • no pattern between amount of estrogen added & reading
  • high standard deviation
  • think that there is a problem with the transformation
  • will try another transformation

yo this is cool