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Subsequently to troubleshooting for both the RFP construct and Uppsala’s chromoproteins, Lambert iGEM ordered chromoproteins from ATUM: Tinsel Purple, Scrooge Orange, and Virginia Violet. While the two purple chromoproteins (Tinsel Purple and Virginia Violet) gave a variance of the color, Scrooge Orange 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. | Subsequently to troubleshooting for both the RFP construct and Uppsala’s chromoproteins, Lambert iGEM ordered chromoproteins from ATUM: Tinsel Purple, Scrooge Orange, and Virginia Violet. While the two purple chromoproteins (Tinsel Purple and Virginia Violet) gave a variance of the color, Scrooge Orange 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. | ||
<br> | <br> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/0/0e/T--Lambert_GA--ATUMjhasbdjhabgd.png" width="400px"> | + | <center><img src="https://static.igem.org/mediawiki/2017/0/0e/T--Lambert_GA--ATUMjhasbdjhabgd.png" width="400px"><br> |
<i style="font-size: 16px; color: white;">Succesful Transformations of ATUM chromoproteins (Scrooge Orange and Tinsel Purple)</i></center> | <i style="font-size: 16px; color: white;">Succesful Transformations of ATUM chromoproteins (Scrooge Orange and Tinsel Purple)</i></center> | ||
Additionally, the 2017 Lambert iGEM team continued attempting a similar construct to the 2016 Lambert iGEM team: p-Lambda-R LacI tsPurple/tsPurple DAS/tsPurple LAA 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. | Additionally, the 2017 Lambert iGEM team continued attempting a similar construct to the 2016 Lambert iGEM team: p-Lambda-R LacI tsPurple/tsPurple DAS/tsPurple LAA 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. |
Revision as of 21:47, 1 November 2017
Experiments
2017 Wetlab 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 p-Lambda-R 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 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 troubleshooting for both the RFP construct and Uppsala’s chromoproteins, Lambert iGEM ordered chromoproteins from ATUM: Tinsel Purple, Scrooge Orange, and Virginia Violet. While the two purple chromoproteins (Tinsel Purple and Virginia Violet) gave a variance of the color, Scrooge Orange 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.
Succesful Transformations of ATUM chromoproteins (Scrooge Orange and Tinsel Purple)
For the reporters alone, sequencing results showed that both tsPurple and tsPurple LAA had the correct part length, but tsPurple DAS 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 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)
2. Nanodrop
3. Digest
4. Gel
5. Ligation
6. Transformation, Plate
7. Colony PCR
8. Gel
9. Inoculate correct colony to liquid culture
In the Lab
Our team members hard at work in the lab.