Difference between revisions of "Team:Carnegie Mellon/Notebook"
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<font color = "orange"><b><u>Goal:</u></b></font> To compare the fluorescence of J23100 RFP with J23115 RFP. | <font color = "orange"><b><u>Goal:</u></b></font> To compare the fluorescence of J23100 RFP with J23115 RFP. | ||
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<li>J23100 RFP did not show consistent results from previous data, mostly likely due to reason mentioned above. New cultures of J23100 RFP and J23115 RFP from newly streaked plates will be used to set up new cultures for measuring. </li> | <li>J23100 RFP did not show consistent results from previous data, mostly likely due to reason mentioned above. New cultures of J23100 RFP and J23115 RFP from newly streaked plates will be used to set up new cultures for measuring. </li> | ||
<li>Re-tested the fluorescence of the J23115 RFP and J23100 since the first set of data from the previous day did not consist of biological replicates. However, the J23100 culture did not seem to grow well since the culture was not red. One reason could be that the plates from which the colonies were taken from were from 6.29.15 and not from the newer plate. This would explain why the fluorescence for J23100 RFP is much lower than expected, when it should be much higher than that for J23115 RFP. </li> | <li>Re-tested the fluorescence of the J23115 RFP and J23100 since the first set of data from the previous day did not consist of biological replicates. However, the J23100 culture did not seem to grow well since the culture was not red. One reason could be that the plates from which the colonies were taken from were from 6.29.15 and not from the newer plate. This would explain why the fluorescence for J23100 RFP is much lower than expected, when it should be much higher than that for J23115 RFP. </li> | ||
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<p> <div class = "dateheader"> 8.3.15 </div></p> | <p> <div class = "dateheader"> 8.3.15 </div></p> | ||
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<li>Started three 2 ml overnight cultures, each consisting of 2 ul of chlorophenical, for J23100 RFP and J23115 RFP </li> | <li>Started three 2 ml overnight cultures, each consisting of 2 ul of chlorophenical, for J23100 RFP and J23115 RFP </li> | ||
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<p> <div class = "dateheader"> 8.4.15 </div></p> | <p> <div class = "dateheader"> 8.4.15 </div></p> | ||
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<li>Measured fluorescence for each overnight culture that was set up the day before (J23100 RFP, J23115 RFP, and empty MACH cells). These values are considered biological replicates. </li> | <li>Measured fluorescence for each overnight culture that was set up the day before (J23100 RFP, J23115 RFP, and empty MACH cells). These values are considered biological replicates. </li> |
Revision as of 16:59, 4 September 2015
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.
Take me to Tab 2 Take me to Tab 3
- First day of work!
- Familiarized ourselves with the lab.
- Made LB (protocol) & poured plates (protocol).
The light fluoresces! We have transformants & isolated proteins.
- 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
- 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
- His-tagged soluble protein extraction (protocol in the Protocol Page ) using a washing solution with dilute imidazole
- Used 50 uL of beads for each
- extracted proteins from 1.5 mL of each culture
- Performed elution from beads
- Used 100 mM EDTA / 10 mM Tris solution to elute proteins from beads
- incubated for 5 minutes
- removed supernatant and repeat for a second elution
- Quantified solution with NanoDrop
- OD of cultures was 0.356 in a 1:10 dilution, thus 4.275 * 10^9 bacteria in each 1.5mL sample.
- Total amount of protein: 395.92 µg
- The elution used before the EDTA elution did not work -- had a pH of 5.0 & needed a pH of 4.5.
- YFP had trouple growing & flourescing.
- Linked is a picture of the trouble with the YFP plates we've made so farLink of the three YFP plates & description.
Concentration of Eluted mRFP | |
First Elution | Second Elution |
6.15 mg/mL | 1.6 mg/mL |
6.353 mg/mL | 1.777 mg/mL |
6.352 mg/mL | 1.523 mg.mL |
Mean: 6.285 mg/mL | Mean: 1.63333 mg/mL |
0.11691 mg/mL | 0.13024 mg/mL |
- Carried out His-tagged soluble protein extraction as normal and then proceeded to elute 2 samples (50 uL of bead solution with protein from 1.5 mL of culture).
- One bead sample eluted three times with 5 min. incubation period in 250 µL of 100 mM EDTA / 10 mM Tris solution for each elution
- Second sample eluted twice with 10 min. incubation period in 250 µL of 100 mM EDTA / 10 mM Tris solution for each elution
- For five minute elutions, first elution had protein concentration of 0.95mg/mL, second elution had 0.355mg/mL, and the third had 1.004 mg/mL.
- For ten minute elutions, the first elution had 0.615 mg/mL concentration and the second elution had 0.328 mg/mL concentration.
- In total, the 5 minute elution had 384.83 µg and the 10 minute elution had 235.75 µg per 1.5 mL culture.
- Ran gel from yesterday's elutions. Link to results here.
- Carried out His-tagged soluble protein extraction and eluted 2 samples (50 uL of bead solution with protein from 1.5 mL of culture, 2 cultures of OFP transformed E. coli, and another 2 of mRFP transformed E. coli).
- One bead sample was eluted three times with 5 min. incubation period in 25 µL of 100 mM EDTA / 10 mM Tris solution for each elution
- Second bead sample was eluted twice with a 10 min. incubation period in 50 µL of 100 mM EDTA / 10 mM Tris solution for each elution
- Eluted proteins quantified using a NanoDrop spectrophotometer.
- Total amount of protein: 25 µL elution mRFP: 56.3235 µg, 50 µL elution mRFP: 81.275 µg, 25 µL elution OFP: 53.6605 µg, 50 µL elution OFP: 142.4135 µg
- Performed his-tagged soluble protein extraction on 4 1.5 mL cultures transformed to produce mRFP, and 4 transformed to produce OFP.
- Elution with 100 mM EDTA / 10 mM Tris solution for 50, 70, 85, and 100 µL volumes for each elution, 3 rounds of elution for each sample.
- Finally have a plate with expression of all 5 fluorescent proteins. insert picture here
- Standard His-tag soluble protein extraction onto 50 μL of Ni-NTA bead solution
- 3 elutions using 5 min incubation time in 50 μL of EDTA buffer
- The concentration of 1 μg/mL that we used for the BSA standard curve did not reveal any relevant information. In the future we will use standards ranging from 3 ug/mL to 20 ug/mL to encompass our data.
- The procedure followed can be found in the Protocols page insert protocol link here.
- See 6/16/2015 for the protocol on BSA standards and testing protein concentration with the Bio-Rad Protein Assay. Today we used the range 3 μg/mL to 20 μg/mL as opposed to 1-10 which we used on 6/16.
- Calculated that we have 2.543 mg of mRFP protein in 1.5mL culture.
- Transformed cells from before did not work, performed transformations again.
- 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 ).
- 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
- 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 Temperature 37°C Sample 108A 108C 109A 109C 111A 111B Reading 5932 6139 6050 5789 5589 6109
- 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
- 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.
- 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.
- 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.
- Forgot to do ligation and transformation, therefore we will redo this tomorrow.
- 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.6.10.15Goal: 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.
6.11.15Goal: 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.
6.12.15Goal: 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 46.15.15Goal: 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.
6.16.15Goal: 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.
6.17.15Goal: 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.
6.18.15Goal: 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 56.23.15Goal: 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.
6.24.15Goal: 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
- Tested the following amounts of estrogen:
- Got very varied and inconsistent results
- Think that there is a problem with the transformation
- Will try another transformation
- Tried the restreaked cells with 1:100,000, 1:1,000,000, and 1:10,000,000 estrogen dilutions
- High standard deviation amongst duplicates, no pattern between amount of estrogen added & reading
- Determined that restreaked cells do not work
- Transformation 4 of Mach cells did not work
- Gave unexpected results with high standard deviation
- Did not seem to be a difference between the different amounts of estrogen
- For tomorrow:
- Made dilutions (1:100) from the starter cultures for the estrogen sensors and controls.
- Set up transformations for CD-cel Gaussia (on plates and in culture) for testing of luciferase activity in response to addition of coelenterazine.
- The isolation of CD-cel domain and gaussia domain didn’t work.
- Started overnight cultures of the Sensor 6 cells from the restreaked plate
- Did not add estrogen
- Each overnight tube from a different colony
- For the next overnight: will add 100 µL of the overnights into 2mL of fresh media & add different dilutions of estrogen then
- Goal: want to see if different colonies gave consistent results
- Made another set of starter cultures for estrogen sensor (with and without estrogen) and controls by adding a colony of each sample into 2 ml of fresh LB media. Cultures shook overnight in 37C shaker.
- Added 20 ul of starter culture to 2 ml of fresh LB containing 2 ul of chlorophenical and 1.2 ul of kanamyacin. This was done three times for each sample. For the J23100 RFP and PelB Gaussia, however, no kanamyacin was added
- 2ml cultures consisting of 2 ul of chloramphenicol were made for J23100 RFP and J23115 RFP and left overnight in the 37C shaker.
- J23100 RFP did not show consistent results from previous data, mostly likely due to reason mentioned above. New cultures of J23100 RFP and J23115 RFP from newly streaked plates will be used to set up new cultures for measuring.
- Re-tested the fluorescence of the J23115 RFP and J23100 since the first set of data from the previous day did not consist of biological replicates. However, the J23100 culture did not seem to grow well since the culture was not red. One reason could be that the plates from which the colonies were taken from were from 6.29.15 and not from the newer plate. This would explain why the fluorescence for J23100 RFP is much lower than expected, when it should be much higher than that for J23115 RFP.
- Started three 2 ml overnight cultures, each consisting of 2 ul of chlorophenical, for J23100 RFP and J23115 RFP
- One 2ml overnight culture without chlorophenical was also made for empty MACH cells as a control.
- Measured fluorescence for each overnight culture that was set up the day before (J23100 RFP, J23115 RFP, and empty MACH cells). These values are considered biological replicates.
- Measured each sample three times (technical replicates) Conclusion: The fluorescence of the J23100 RFP was 5 times larger than that of the J23115 RFP.
- Tried to get similar-sized colonies
- 100 uM, 20 uM, 10 uM, 1 uM, 100 nM, 10 nM, 1 nM, 0 nM
- No pattern between amount of estrogen added & reading
- High standard deviation
- Do another transformation -- possibly get Donna & Michelle to do it
- Try again with the colonies we have now
- Want to see if lower dilutions of estrogen give lower readings
The Arduino is a piece of programmable hardware created by several graduate students in Italy in the early 2000’s. The name itself comes from a bar they used to hang out in, which in turn was named after an old Italian king, Arduin of Ivrea. It was created because the hardware they had previously been using was $100 and they knew some people couldn’t afford that, so they designed a cheaper alternative. This is the embodiment of the Maker’s Movement Era!
And with that, the Arduino was born. The model we use, the “Arduino Uno,” contains a programmable chip that we can code via USB, and it has fourteen digital pins along with eight analog pins that can be used to communicate with a circuit based on voltage levels. With this, the possibilities are endless! People have made remote control lawnmowers and quadcopters, they’ve built robots and video games, and they’ve even made sensors and alarms to be used for home security and ease of access! This is what the Arduino can do: bring easy circuit making to anyone’s door step to help improve quality of life.