Team:Lambert GA/Experiments


Experiments


2017 Wet Lab Summary


The 2017 Lambert iGEM team worked two-fold on developing a proof of concept for the Chrome-Q and assembling a genetic construct to use the Chrome-Q to quantify data. In the process of advancing the Chrome-Q, there were three methods in which Lambert iGEM attempted to grow E. coli expressing color for data collection.

Several weeks were devoted to construct pλR LacI R011 RFP in the NEB 10-beta E. coli strain, with individual parts given from Monica McNerney at the Georgia Institute of Technology. However, the lack of RFP expression in the cells led the team to believe that the sequences - particularly RFP - were mutated when attempting to digest and ligate the parts together.

Next, the team referred to the parts registry to use existing chromoproteins. From Uppsala 2013, Lambert iGEM attempted to ligate AmilCP (BBa_K592009), Amaj Lime (BBa_K1033916), and CJ Blue (BBa_K592011) into backbones to obtain a variety of measurements on the Chrome-Q using the HSV color space. After adjusting digestion, ligation, and transformation protocols and still failing to see the expected color expression, the team referred back to Stanford-Brown’s wiki page on their usage of the chromoproteins for their project in 2016. It was discovered that their team also did not successfully transform those chromoproteins; as a result, they had ordered their parts to obtain their results.


Subsequently, to troubleshoot for both the RFP construct and Uppsala’s chromoproteins, Lambert iGEM ordered chromoproteins from ATUM: TinselPurple, ScroogeOrange, and VirginiaViolet. While the two purple chromoproteins (TinselPurple and VirginiaViolet) gave a variance of the color, ScroogeOrange provided a contrasting pigment for the Chrome-Q to measure. After successful transformations, liquid cultures were induced with varying levels of IPTG - 0uM, 10uM, 100uM, 500uM, and 1mM. Distinct visual differences were observed: incremental concentrations of IPTG directly correlated with an increase in chromoprotein expression. Measurements were made on the spectrophotometer and the Chrome-Q for comparison.



Successful Transformations of ATUM chromoproteins (ScroogeOrange and TinselPurple)

Additionally, the 2017 Lambert iGEM team continued attempting a similar construct to the 2016 Lambert iGEM team: pλR LacI tsPurple/tsPurpleDAS/tsPurpleLAA pLac ClpXP CI. DAS (a moderate degradation tag) and LAA (a strong degradation tag) allowed rates of degradation to be compared when measuring the RGB values of the cells on the Chrome-Q. The promoter, pλR LacI, had very low miniprep concentrations, and it was hypothesized that the toxicity to the cells prevented from both transformation and miniprep efficiencies to be proficient for the rest of the assemblies. As a result, the team switched to R0040, a promoter that was transformed from this year’s InterLab study. This was successfully ligated to the three reporters, but the colonies did not turn purple when transformed in the NEB 5-alpha E. coli strain and the Keio strains.

For the reporters alone, sequencing results showed that both tsPurple and tsPurpleLAA had the correct part length, but tsPurpleDAS did not include the degradation tag in its sequencing result. As a result, the team contributed these new composite parts to the iGEM registry.

The final third of the construct - pLac ClpXP CI - was successfully assembled and sent for sequencing, which revealed that CI had been mutated during assembly and therefore not functional. Due to time constraints, the 2017 Lambert iGEM team did not have time to put together the entire construct.

While characterizing non-lysosomal inducible protein degradation, the 2017 Lambert iGEM team developed the Chrome-Q to quantify the degradation of different chromoproteins.

Workflow


1. Miniprep/Nanodrop
2. Digest
3. Gel
4. Ligation
5. Transformation, Plate
6. Colony PCR (Screening)
7. Gel
8. Inoculate correct colony to a liquid culture.

Materials:


Miniprep: grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips

Nanodrop: nanodrop machine, miniprepped DNA, Kimtech wipes, micropipette and tips

Digest: miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips

Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

Ligation: vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips

Transformation: ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips Plate: agar plate, micropipette and tips, beads

Colony PCR: dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice

Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

Inoculate: LB media, dilution, micropipette and tips


Protocol:


1. Miniprep (using Omega protocol)
1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube.
1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media. (Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.)
1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.
1.3 Add 250uL of Solution I mixed with RNase A (pre-added). Vortex to mix thoroughly. Transfer the suspension into a new 1.5mL microcentrifuge tube.
1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate.
1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes!
1.5 Add 350uL of Solution III. Invert several times until a white precipitate forms. Centrifuge at 13,000xg or 17,900rcf for 10 minutes. A compact white pellet should form at the bottom of the tube.
1.6 Insert a mini column into a 2mL collection tube.
1.7 Transfer the clear supernatant into the mini column using a micropipette. Centrifuge at the maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.
1.7.1 Be careful not to get any parts of the pellet! Tilt at an angle with the pellet at the top when micropipetting is advisable.
1.7.2 Think about what you are discarding versus what you want to keep!
1.8 Add 500uL of the HBC Wash Buffer diluted in isopropanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.
1.8.1 All wash buffers will be centrifuged for 1 minute.
1.9 Add 700uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.
1.10 Centrifuge the empty mini column at the maximum speed (13,000xg) for 2 minutes to remove the ethanol.
1.11 Transfer the mini column to a nuclease-free 1.5mL microcentrifuge tube.
1.12 Add 50uL of Elution Buffer (or sterile deionized water). Let it sit in room temperature for 60 seconds. Centrifuge at maximum speed (13,000xg) for 60 seconds.
1.13 Store eluted DNA at -20℃.



2. Nanodrop
2.1 Vortex before nanodrop.
2.2 Wipe down the nanodrop machine with Kimtech wipes to make it sterile.
2.3 Set the program to analyze nucleic acids [because you are dealing with plasmid DNA].
2.4 Do a blank test to ensure that the platform is sterile.
2.5 Load 1uL of the miniprepped DNA onto the platform.
2.5.1 (Have steady hands. The sample needs to be in the center for best results.)
2.6 Click “measure” on the nanodrop for analysis.
2.7 Write down measurements for the concentration of DNA (in ng/uL), A260, A280, 260/280 (should be around 1.8), and 260/230 (should be around 2.1).

3. Digest
3.1 Dilute up to 1ug DNA to 17uL with dH₂O.
3.1.1 Take concentration of DNA from nanodrop and convert from ng/uL to ug/uL. Next, set up a proportion to find out how many uL you need to get 1 ug of DNA.
3.1.2 20uL (total reaction) - 2uL RE-Mix - 1uL standard enzyme = uL dH₂O
3.2 Use a microcentrifuge tube to put the reaction in. Put in the contents in this order: water, DNA, enzymes.
3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme.
  3.2.1.1 E and X = 10X RE-Mix
  3.2.1.2 S and P = standard enzymes
3.3 Incubate at 37℃ for 1 hour for standard enzymes, then at 80℃ for deactivation.

4. Gel
4.1 Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the DNA runs to red.
4.2 Pour the TAE buffer evenly to completely cover the gel.
4.3 Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA to be put in each well.
4.4 Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes.
4.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.

5. Ligation
5.1 Use Antarctic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure. Make calculations using a 3:1 molar ratio of insert to backbone. Refer to the two tables below.
5.2 Put in each component in a microcentrifuge tube while on ice. They should be pipetted into the tube in this order: water, DNA, ligase buffer, ligase.
5.3 The ligase buffer should be thawed and resuspended at room temperature.
5.3.1 Gently mix by pipetting up and down and microfuge briefly.
5.4 Incubate at room temperature for 1 hour at 37℃

6. Transformation, Plate
6.1 Thaw materials on ice for 5 minutes.
6.2 Put 10uL of ligation mixture into 100uL competent cells in a microcentrifuge tube.
6.3 Flick the tube to mix.
6.4 Put on ice for 30 minutes.
6.5 Add 200uL of LB media.
6.6 Incubate at 37℃ for one hour.
6.7 Plate 150uL of cells onto a plate. Make sure plate has the correct antibiotic (based on vector backbone)! Grow overnight.

7. Colony PCR
7.1 Pick colonies with a combination of phenotypes i.e. large/small, red/white. Dilute each colony in 40uL dH₂O, 1uL DNA from ligation if transformation is successful.
7.1.1 If necessary, do a quick spin to make sure all the liquid is at the bottom.
7.2 Make the following master mix on ice in this order: 63uL dH₂O, 20uL buffer, 5uL VF₂ primer, 5uL VR primer, 2uL dNTP, 1uL Q5 polymerase.
7.3 Aliquot the master mixes into PCR tubes, then add 1uL of the DNA dilution.
7.3.1 Make sure PCR tubes are labeled properly and carefully!
7.4 Transfer the PCR tubes to a PCR machine and begin thermocycling.
7.4.1 Initial Denaturation: 98℃ for 30 seconds
7.4.2 25-35 Cycles: 98℃ for 5-10 seconds, 50-72℃ for 10-30 seconds, 72℃ for 20-30 seconds/kb
7.4.3 Final Extension: 72℃ for 2 minutes
7.4.4 Hold 4-10℃
 
8. Gel
8.1 Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the PCR samples run to red.
8.2 Pour the TAE buffer evenly to completely cover the gel.
8.3 Using a micropipette, put 3uL of PCR samples in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more PCR sample to be put in each well.
8.4 Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes.
8.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.

9. Inoculate correct colony to liquid culture
9.1 Get the remaining 39uL of colony dilution.
9.2 Get LB media and make sure to use the appropriate antibiotic resistance.
9.3 Mix the colony dilution into the media.
9.4 Grow overnight.

Secret Photo 2


In the Lab



Our team members hard at work in the lab.

Software


Chrome-Q App

Chrome-Q is a Xamarin C# app created in Microsoft Visual Studio 2017 Community Edition. The app was developed for mobile Android devices and will soon be available for iOS devices. A photo is taken (within the app) of the Chrome-Q dome target base (that contains the rows of samples in triplicate). The app finds the samples in the photo by looking for low luminance values compared to the high luminance values in the white background. Then, the app finds the average hue and luminance for each sample by averaging the RGB values for all of the pixels in the circular sample. It groups the samples into rows by comparing their vertical locations in the photo. After grouping into rows, it calculates the average RGB values for the entire row to generate an average hue and luminance for the triplicate. The average hue and luminance are utilized to calculate the standard deviation of the triplicate. By looking at the standard deviation, it was determined that the hue values were most consistent in the triplicate. These values can then be used to compare relative levels of degradation between the different constructs (tsPurple, tsPurpleDAS, and tsPurpleLAA). Chrome-Q system methodology can be found in the Model page.





Chromoprotein samples in the Chrome-Q well plate and the Chrome-Q app recognizing the colors (represented by the green dots)



Data gathered on tsPurple samples using the Chrome-Q model and app



The hue values of tsPurple at different levels of IPTG induction

Important Note: Hues values range from 0 to 360 degrees. The hue values in the samples begin at around 270 degrees (purple) and increase through 360 degrees. The hues value 0 uL IPTG concentration wraps around to a low hue value of around 40 degrees. To process these hues, 360 degrees were added to values less than 180 degrees, which is why some values are greater than 360.





The % purple of tsPurple samples at different levels of IPTG induction



Team member Emily Gibson using the Chrome-Q system


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