iGEM 2017 — Team Lambert_GA

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# Team:Lambert GA/Notebook

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- [Project](https://2017.igem.org/Team%3ALambert_GA/Description)

  [Description](https://2017.igem.org/Team%3ALambert_GA/Description)
  [Design](https://2017.igem.org/Team%3ALambert_GA/Design)
  [Experiments](https://2017.igem.org/Team%3ALambert_GA/Experiments)
  [Notebook](https://2017.igem.org/Team%3ALambert_GA/Notebook)
  [InterLab](https://2017.igem.org/Team%3ALambert_GA/InterLab)
  [Model](https://2017.igem.org/Team%3ALambert_GA/Model)
  [Results](https://2017.igem.org/Team%3ALambert_GA/Results)[Demonstrate](https://2017.igem.org/Team%3ALambert_GA/Demonstrate)
  [Attributions](https://2017.igem.org/Team%3ALambert_GA/Attributions)
- [Parts](https://2017.igem.org/Team%3ALambert_GA/Part%20Collection)

  [Part Collection](https://2017.igem.org/Team%3ALambert_GA/Part%20Collection)
  [Updated Parts](https://2017.igem.org/Team%3ALambert_GA/Updated_Part)
  [Composite Parts](https://2017.igem.org/Team%3ALambert_GA/Composite%20Part)
[Safety](https://2017.igem.org/Team%3ALambert_GA/Safety)- [Human Practices](https://2017.igem.org/Team%3ALambert_GA/HP/Silver)

  [Silver HP](https://2017.igem.org/Team%3ALambert_GA/HP/Silver)
  [Integrated and Gold](https://2017.igem.org/Team%3ALambert_GA/HP/Gold_Integrated)
  [Public Engagement](https://2017.igem.org/Team%3ALambert_GA/Engagement)
- [Awards](https://2017.igem.org/Team%3ALambert_GA/Hardware)

  [Applied Design](https://2017.igem.org/Team%3ALambert_GA/Applied_Design)
  [Hardware](https://2017.igem.org/Team%3ALambert_GA/Hardware)
  [Model](https://2017.igem.org/Team%3ALambert_GA/Model)
  [Software](https://2017.igem.org/Team%3ALambert_GA/Software)
[Judging Form](https://igem.org/2017_Judging_Form?team=Lambert_GA)

# **Notebook**

![](https://static.igem.org/mediawiki/2017/b/bc/T--Lambert_GA--purpleline.png)

Week 1 (Aug 3):

Since we had been having difficulties working with pλR LacI, we discussed the merits of using a different promoter for our tsPurple constructs.

Week 2 (Aug 7):

After the previous week's discussions, we transformed, inoculated, and miniprepped weak pLac (hoping that a weaker promoter would cause less strain on our cells). In addition, we attempted to ligate the tsPurples into 1C3, however the miniprep concentrations had been too low, so we needed to order more of our parts from IDT.

 Week 3 (Aug 14):

We came to the decision that pLac would not work when our construct was fully assembled because it is the same promoter that is on the second part of our construct. Due to this, we moved back to pλR LacI and inoculated liquid cultures from a pre-existing plate. In addition, we transformed and then miniprepped our tsPurple constructs, however the concentrations of both pλR LacI and the tsPurples were shockingly low so we decided to run a PCR next week.

Week 4 (Aug 21):

After last week's low miniprep concentrations we ran PCRs of the tsPurples, pλR LacI, and pLac-ClpXP-CI. We also discussed our lab work with Dr. Styczynski and Monica McNerney to determine potential sources of error.

Week 5 (Aug 28):

After last week's PCR we did a cleanup and nanodropped, which yielded good concentrations. We then sent pλR LacI and pLac ClpXP CI for sequencing and ligated and transformed our tsPurple constructs.

Week 6 (Sep 4):
Since the previous transformations were unsuccessful (with concerns about contamination and plates with the wrong antibiotic), we decided to re-transform. Since those were successful, we ran a colony PCR, however the gel showed that the colony PCR was unsuccessful. Meanwhile, we transformed our RFP proof of concepts.

Week 7 (Sep 11):
We inoculated liquid cultures of pλR LacI and retransformed our tsPurple constructs after the unsuccessful colony PCR, however the transformations were unsuccessful as well so we decided to return to earlier minipreps and start over. After starting over and retransforming, it turned out third time is not the charm since it was unsuccessful again. We decided to go another step back and re-digest from the original hydrated tsPurple constructs. Simultaneously, we had an unsuccessful miniprep of pλR LacI (but a successful one of pLac-ClpXP-CI), so we went back to an old digest and ran all three tsPurple digests and pλR LacI on a gel. While the tsPurples were the expected base pair length, pλR LacI did not show up, potentially because of incredibly low concentration.

Week 8 (Sep 18):
After realizing some calculations were off the previous week, we re-inoculated from old transformations. In the mean time, we hydrated amilCP, amajLime, and cjBlue from IDT.

Week 9 (Sep 25):
Our county had fall break this week and we were unfortunately unable to access our lab space, which put us back a week in our lab flow. In this time, some of our team members drove to UGA's lab so that we could complete the InterLab study.

Week 10 (Oct 2):
We picked up where we left off before fall break and ligated pλR LacI with tsPurple, tsPurpleDAS, and tsPurpleLAA. Unfortunately, when we transformed they were unsuccessful, so we went back and re-digested the separate parts.

Week 11 (Oct 9):
In order to troubleshoot our pλR LacI construct, we decided to attempt to recreate the part from the iGEM Distribution Kits rather than our previous stocks. We utilized pλR, an RBS, and LacI that was present within the Kit Plates as listed on the Parts Registry, and navigated through the cloning workflow to build the part.

Week 12 (Oct 16):
After running the digests of our finished constructs on a gel, we realized that the pλR LacI was not the correct length. We retried digestion using BSA to stabilize the digestion, but it was still unsuccessful. We had already performed the ligation of pλR LacI during the digest to save time, but safely discarded the ligations after realizing it was unsuccessful. We also prepared and sent R0011 ClpXP CI for sequencing.

Week 13 (Oct 23):
We ligated the tsPurple into the backbones for parts submission, using pSB1C3 as the plasmid backbone. The DNA was additionally dried down and shipped. We also transformed purchased plasmids for color expression and miniprepped them. For the construction of the construct, we obtained the promoter from the InterLab Study Negative Control (R0040) but mislabeled it in the lab notebook due to an apparent change in parts list sent to us. We also miniprepped R0011 ClpXP CI again to increase our stock amounts. We digested the promoter and tsPurples for eventual ligation.

Week 14 (Oct 30):
We ligated the R0040 promoter to the tsPurples and transformed them into the Keio Strains as well as DH5. We also sent our tsPurples in for sequencing.

## View Our Cloning Workflow

Click here to download your own [Ligation Calculator](https://static.igem.org/mediawiki/2017/a/a5/Copy_of_Ligation_Calculator.xlsx).

Cloning Workflow

*Cloning Workflow*

## View Our Team's Notebooks

Team Notebook

*Team Notebook*

Team Notebook

*Natalie Shih's Notebook*

Team Notebook

*Juniors' Lab Notebook*

[![](https://static.igem.org/mediawiki/2017/3/36/RevisedLambertLonghorn.png)](http://www.forsyth.k12.ga.us/lhs)

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# Team:Lambert GA/Experiments

[Home](https://2017.igem.org/Team%3ALambert_GA)- [Team](https://2017.igem.org/Team%3ALambert_GA/Team)

  [Team](https://2017.igem.org/Team%3ALambert_GA/Team)
  [Collaborations](https://2017.igem.org/Team%3ALambert_GA/Collaborations)
- [Project](https://2017.igem.org/Team%3ALambert_GA/Description)

  [Description](https://2017.igem.org/Team%3ALambert_GA/Description)
  [Design](https://2017.igem.org/Team%3ALambert_GA/Design)
  [Experiments](https://2017.igem.org/Team%3ALambert_GA/Experiments)
  [Notebook](https://2017.igem.org/Team%3ALambert_GA/Notebook)
  [InterLab](https://2017.igem.org/Team%3ALambert_GA/InterLab)
  [Model](https://2017.igem.org/Team%3ALambert_GA/Model)
  [Results](https://2017.igem.org/Team%3ALambert_GA/Results)[Demonstrate](https://2017.igem.org/Team%3ALambert_GA/Demonstrate)
  [Attributions](https://2017.igem.org/Team%3ALambert_GA/Attributions)
- [Parts](https://2017.igem.org/Team%3ALambert_GA/Part%20Collection)

  [Part Collection](https://2017.igem.org/Team%3ALambert_GA/Part%20Collection)
  [Updated Parts](https://2017.igem.org/Team%3ALambert_GA/Updated_Part)
  [Composite Parts](https://2017.igem.org/Team%3ALambert_GA/Composite%20Part)
[Safety](https://2017.igem.org/Team%3ALambert_GA/Safety)- [Human Practices](https://2017.igem.org/Team%3ALambert_GA/HP/Silver)

  [Silver HP](https://2017.igem.org/Team%3ALambert_GA/HP/Silver)
  [Integrated and Gold](https://2017.igem.org/Team%3ALambert_GA/HP/Gold_Integrated)
  [Public Engagement](https://2017.igem.org/Team%3ALambert_GA/Engagement)
- [Awards](https://2017.igem.org/Team%3ALambert_GA/Hardware)

  [Applied Design](https://2017.igem.org/Team%3ALambert_GA/Applied_Design)
  [Hardware](https://2017.igem.org/Team%3ALambert_GA/Hardware)
  [Model](https://2017.igem.org/Team%3ALambert_GA/Model)
  [Software](https://2017.igem.org/Team%3ALambert_GA/Software)
[Judging Form](https://igem.org/2017_Judging_Form?team=Lambert_GA)

# **Experiments**

![](https://static.igem.org/mediawiki/2017/b/bc/T--Lambert_GA--purpleline.png)

## 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.

![](https://static.igem.org/mediawiki/2017/0/0e/T--Lambert_GA--ATUMjhasbdjhabgd.png)
*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](https://static.igem.org/mediawiki/2017/c/ca/Huuuuuuuuuulalahoho.jpeg)

## In the Lab

![](https://static.igem.org/mediawiki/2017/7/76/T--Lambert_GA--WetLab1.jpeg)
![](https://static.igem.org/mediawiki/2017/1/18/T--Lambert_GA--WetLab2.jpeg)
![](https://static.igem.org/mediawiki/2017/3/38/Alyssalabexp.jpeg)

 *Our team members hard at work in the lab.*

# **Software**

![](https://static.igem.org/mediawiki/2017/b/bc/T--Lambert_GA--purpleline.png)

### 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.

![](https://static.igem.org/mediawiki/2017/1/1c/Appfindingcircles.jpeg)

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

![](https://static.igem.org/mediawiki/2017/b/bb/Newtspurplechart.png)

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

![](https://static.igem.org/mediawiki/2017/1/11/Updatedhuetspurplechart.png)

 *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.

![](https://static.igem.org/mediawiki/2017/a/a0/%25purplechart.png)

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

![](https://static.igem.org/mediawiki/2017/6/62/Notjankychromeqpic.jpeg)

 *Team member Emily Gibson using the Chrome-Q system*

[![](https://static.igem.org/mediawiki/2017/3/36/RevisedLambertLonghorn.png)](http://www.forsyth.k12.ga.us/lhs)

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# Team:Lambert GA/Results

[Home](https://2017.igem.org/Team%3ALambert_GA)- [Team](https://2017.igem.org/Team%3ALambert_GA/Team)

  [Team](https://2017.igem.org/Team%3ALambert_GA/Team)
  [Collaborations](https://2017.igem.org/Team%3ALambert_GA/Collaborations)
- [Project](https://2017.igem.org/Team%3ALambert_GA/Description)

  [Description](https://2017.igem.org/Team%3ALambert_GA/Description)
  [Design](https://2017.igem.org/Team%3ALambert_GA/Design)
  [Experiments](https://2017.igem.org/Team%3ALambert_GA/Experiments)
  [Notebook](https://2017.igem.org/Team%3ALambert_GA/Notebook)
  [InterLab](https://2017.igem.org/Team%3ALambert_GA/InterLab)
  [Model](https://2017.igem.org/Team%3ALambert_GA/Model)
  [Results](https://2017.igem.org/Team%3ALambert_GA/Results)[Demonstrate](https://2017.igem.org/Team%3ALambert_GA/Demonstrate)
  [Attributions](https://2017.igem.org/Team%3ALambert_GA/Attributions)
- [Parts](https://2017.igem.org/Team%3ALambert_GA/Part%20Collection)

  [Part Collection](https://2017.igem.org/Team%3ALambert_GA/Part%20Collection)
  [Updated Parts](https://2017.igem.org/Team%3ALambert_GA/Updated_Part)
  [Composite Parts](https://2017.igem.org/Team%3ALambert_GA/Composite%20Part)
[Safety](https://2017.igem.org/Team%3ALambert_GA/Safety)- [Human Practices](https://2017.igem.org/Team%3ALambert_GA/HP/Silver)

  [Silver HP](https://2017.igem.org/Team%3ALambert_GA/HP/Silver)
  [Integrated and Gold](https://2017.igem.org/Team%3ALambert_GA/HP/Gold_Integrated)
  [Public Engagement](https://2017.igem.org/Team%3ALambert_GA/Engagement)
- [Awards](https://2017.igem.org/Team%3ALambert_GA/Hardware)

  [Applied Design](https://2017.igem.org/Team%3ALambert_GA/Applied_Design)
  [Hardware](https://2017.igem.org/Team%3ALambert_GA/Hardware)
  [Model](https://2017.igem.org/Team%3ALambert_GA/Model)
  [Software](https://2017.igem.org/Team%3ALambert_GA/Software)
[Judging Form](https://igem.org/2017_Judging_Form?team=Lambert_GA)

# Results

![](https://static.igem.org/mediawiki/2017/b/bc/T--Lambert_GA--purpleline.png)

## Conclusion and Results

Expected results correlate with the genetic design found on the Experiments page. To recapitulate, the full construct contained the LacI repressor system, which initially impeded the promotion of sequence, ClpXP CI, located downstream of pLac; upon IPTG induction, LacI, when encountered by the chemical, exhibited repression as IPTG molecules bound to Lac repressor proteins. This inhibition allowed promotion of the latter portion of our construct: pLac ClpXP CI. Because of ClpXP’s activation, the transcribed protein was able to recognize the middle portion of our construct containing an SsrA tag attached to tsPurple and, thereby, degrade our reporter chromoprotein. In addition, the CI (competitive inhibitor), was able to bind and inhibit pλR, obstructing the promotion of tsPurple. Therefore, the working construct would supply us with a concentration of translated tsPurple dependant upon the strength of the attached degradation tag (LAA, DAS or no tag present); color expression would then persist inconstancy as the CI repressed the excess promotion of tsPurple. Following the workflow, we then would have transformed our constructs into varied Keio Knockout and Wild strains in order to validate the functional portions of ClpXP. Concluding the institution of our genetic parts into Keio strains, the inoculation of colonies - using the refined methodology demonstrated in our Model and Demonstrate pages- into triplicate trials of 0M, 10 uM, 100 uM, 500 uM, and 1mM concentrations of IPTG, with antibiotic Luria broth as our solvent, would provide us with enriched data to then utilize our Chrome-Q. From calculated HSV color values, it is predicted that as IPTG concentration increase then the hue values will decrease. Hue is measured from 0 to 360 degrees; lower magnitude hue values indicate darker spectrum colors and higher magnitude hue values indicate lighter spectrum colors. Although we are using the HSV color model we decided to omit saturation values. Because saturation measures opacity/ transparency and the data collected will range from a purple to yellow (non-expressive cells) coloration, then calculations of opacity are ineffectual. Finally, value, synonymous to the term “luminance” is also predicted to decrease as IPTG concentrations increase. Luminance indicates emitted light intensity in terms of the amount of light absorbed (low luminance values) and emitted (high luminance values). Because the yellow coloration, observed in non-expressive cells are "brighter" and emit more light than the purple, IPTG induced cells, than it is predicted luminance values will decrease as IPTG levels increase.

As lab work concluded the proposed construct could not be successfully assembled. The initial portion of the construct: pλR LacI, yielded low miniprep concentrations that were impractical to proceed within the cloning workflow. We then attempted to ligate pλR B0034 LacI, but gel results, determining the existence of the sequences, were inconclusive. Because numerous experiences with the promoter, pλR, had not delivered applicable results, the R0040 promoter, obtained from the Interlab Study, was instituted as a replacement. Despite the initial obstacle, the middle parts of the proposed construct, being tsPurple and increasingly potent degradation tags, were ordered from IDT and successfully underwent the cloning procedure. The parts: tsPurple, tsPurple DAS, and tsPuple LAA, were sent for sequencing. The sequence containing LAA, and the sequence without a degradation tag both were verified as complete and void of mutations; the sequence containing DAS was received as lacking the degradation tag. The latter portion of the construct, composed of pLac ClpXP CI, was successfully assembled and sent for sequencing. Results from sequencing concluded that CI had mutated and, therefore, not functional. From the corroborated cloning parts, the sequences containing LAA and no degradation tags were individually ligated to the R0040 promoter and RBS. The ligations were then transformed into competent, NEB 5-Alpha cells, plated and visualized the next day. The presence of purple coloration verified the success of ligation. Despite lacking the full, genetic construct, we managed to obtain viable data from the Chrome-Q system using the proof of concept.

Proof of Concept utilized ATUM’s Protein Paintbox with the chromoproteins: TinselPurple (tsPurple), ScroogeOrange, and VirginiaViolet, and successfully underwent the optimized protocol. The resulting color expression, correlating with incremental concentrations of IPTG, was analyzed using the Chrome-Q system. Methodology for the Chrome-Q system can be found on the Model page. The data obtained was only calculated for tsPpurple induced cells:

Concentration is measured in microliters (uL). Hue is measure from 0 to 360 degrees , and saturation and value are measured from a scale, 0-100. Saturation values were not calculated because the mobile app’s purpose was to accurately calculate color percentage. Because saturation measures opacity/transparency, and the colors visualized from the cells ranged from a yellow hue (0M IPTG) to dark purple (1mM IPTG) then measurements of opacity/ transparency are ineffectual. Luminance is calculated using the HSV color space.

## Analysis of tsPurple Data

As predicted, hue values decreased as IPTG concentrations increased with only slight variation. This variation is found in the 500 uM IPTG samples, which had a value of 100% purple, and is greater than the recorded 99.5% purple value in the 1000 uM IPTG tube. This variation may be an error originating from when pictures were taken using the Chrome-Q. Despite this miscalculation, the 500 uM IPTG was recorded as 100.0% purple because this data point represents the relative percentage of purple against the total set of data. Data representing 100% purple indicates the minimum hue value and data representing 0.0% purple indicates the maximum hue value. (Note: the values where hue equals 400.2 correlates with 40.2 degrees, since values over 360 degrees indicates lighter coloration, such as the “yellow” found in non-expressive E.coli). As for results, the calculated mean was 316.1 uM IPTG, which indicates at what IPTG level the highest percentage of purple will occur. The other values measured were luminance values, which indicated the amount of light emitted or reflected. These results reveal that as IPTG concentrations increase, luminance values decrease. This portion of the data confirmed the expected results since the lighter cell color found in non-expressive “yellow” cells emits a greater light intensity than the purple found in IPTG induced cells. An important point to note is that the values shown in the table and graphs are averages from rows of triplicates to reduce the effects of variability. Overall, the results from tsPurple and the Chrome-Q app indicated a negative correlation between hue and IPTG concentration, however more trials should be completed to further standardize the tsPurple results.

## Scrooge Orange and Virginia Violet

The data collected from the Scrooge Orange and VirginiaViolet trials were congruent with the hypothesis. Hue values for Scrooge Orange, although lesser in intensity than the data calculated for the tsPurple and VirginiaViolet trials, decreased as IPTG values increased. Similarly, luminance values decreased as IPTG concentrations increased along with a reduction of variability in Virginia Violet and ScroogeOrange trials when comparing this data to the tsPurple trials. Standard deviation values demonstrated a relatively low variability throghout the data. There also presented a negative correlation between IPTG concentrations and hue values.

## Conclusion

In order to appropriately characterize our proteolysis, we conducted an additional study to serve as a proof of concept. The ATUM Protein Paintbox allowed us to verify the changes in hue and percent color upon induction with IPTG. The induction of the cells with IPTG induced additional expression of the chromogenic protein due to the inhibition of LacI’s repressor proteins, allowing for T5 to continuously express the chromoprotein. There was direct positive correlation between IPTG concentration and the percent color expressed by the cells, and a direct negative correlation between IPTG concentration and hue. This trend visualized between hue and IPTG can be visualized using the RGB color space. The specific RGB values are quantified in degrees between 0 and 360, and the Hue is calculated using an algorithm we developed. Although the RGB values are visualized in a cylindrical space, the specific Hue value is obtained from a specific point value, allowing the trend to be validated. As IPTG concentration increased, the T5 expression allowed for an increase in the chromogenic protein concentration, leading to an increased expression of color, verifying the increase in percent color. In addition, our usage of both tsPurple and Virginia Violet proved that despite the similarity in color, there is a substantial difference in hue and percent color across induction levels between the two chromoproteins. Through this, we were able to prove the concept of chromoprotein expression across various levels of IPTG Induction.
For the construct we created, consisting of the tsPurple with a constitutive promoter and tsPurple-LAA with a constitutive promoter, we were additionally able to visualize the difference in chromoprotein expression. As the LAA is an SsrA degradation tag, the tsPurple LAA was likely targeted by naturally occurring ClpXP proteins, allowing for the degradation of the purple chromoprotein. This led to almost all purple color being absent in the liquid culture, verifying the occurrence of the protein degradation. On the other hand, the tsPurple with no degradation tag was constitutively expressed, allowing for complete color expression within the cultures. These were analyzed in comparison to regular E. Coli cells, and the hue/percent color values for the tsPurple-LAA were almost analogous to the regular E. Coli cells, while the tsPurple with no degradation tag had high hue and percent color values, indicating the functioning of the ClpXP mechanism occurred successfully.
Through the comparison of our experimental data with our proof of concept, we were able to successfully discern a variation in hue/percent color values with different levels of protein expression and degradation, serving as an effective measurement model. Through the usage of our Chrom-Q prototype, we were able to quantify these variations in values using an alternative model at a low-cost, thereby having the potential to serve as a new basis of measurement in underfunded labs without access to plate readers.
Furthermore, the presence of a negative correlation between IPTG concentrations and luminance/ hue values indicates a quantified trend in chromoprotein expression. The accuracy of the engineered Chrome-Q system is also verified in the differences found in the magnitude of calculated hue and luminance values for each chromoprotein, where tsPurple and VirginiaViolet had the highest mean hues/luminance values, while Scrooge Orange had the lowest mean hue/luminance values. Overall, the Chrome-Q system is a valid system when quantifying and distinguishing chromoprotein color expression.

*Results from analysis of the chromoprotein Scrooge Orange*

*Results from analysis of the chromoprotein Virginia Violet*

*A progression of the Chrome-Q app recognizing the different colored dots*

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