Difference between revisions of "Team:Lambert GA/Hardware"

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       <a href="https://2017.igem.org/Team:Lambert_GA/Parts" class="dropbtn">Parts</a>
 
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<br>
 
<center> <h1 id="MainTitle"><b> Description</b></h1>
 
<img src="https://static.igem.org/mediawiki/2017/b/bc/T--Lambert_GA--purpleline.png" style="width:18%; margin:auto;"></center>
 
 
<br>
 
<br>
  
<section>
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<center> <h1 id="MainTitle"><b> Hardware </b></h1> <img src="https://static.igem.org/mediawiki/2017/2/26/T--LambertGA--purpleline.jpg" style="width:18%; margin:auto;"> </center> <br>
<div class="container">
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<div class="row">
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<div class="col-md-12 col-sm-10">
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The Lambert iGEM team is attempting to address two themes that embody synthetic biology and its diverse range of applications: precision and universality.  Last year, the 2016 Lambert iGEM team attempted to address the issue of overexpression of proteins by devising a “switch”, a genetically engineered construct that degraded GFP (green fluorescent protein) using a protease mechanism ClpXP upon induction of IPTG.  This year’s team is continuing to build upon this idea of characterizing ClpXP by further developing our genetic construct via the use of various chromoproteins and promoters.  The data will be quantified using the ChromQ, a camera device that standardizes the light source to accurately measure the amount of chromoprotein present in a pellet of cells.  Our ChromQ aims to allow especially under-funded labs to have access to a device that quantifies data without spending thousands of dollars; in addition, a functional app and mathematical model are being created to be able to compare expressions of pigments before, during, and after induction of IPTG. Ultimately, the 2017 Lambert iGEM team is striving to further characterize a precise, non-lysosomal induced protein degradation (ClpXP) and to market an affordable device (ChromQ) that can be universally used to quantify data.
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 +
<div class="center">
 +
<img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/c/c4/T--LambertGA--camera.jpg">
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</div><br>
  
<br><br>
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<center><h3 style="color: #D49AE6;">Function</h3></center>
<center><img src="https://static.igem.org/mediawiki/2017/b/b3/T--Lambert_GA--overexpression.png" style="width:70%; margin:auto;"></center>
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<p style="width: 100%; margin: auto; font-size: 16px;">The ChromQ Light Chamber is a 3D-printed imaging measurement system used to quantify results of nutrient deficiency. For our current project, it is also used to quickly and inexpensively measure relative protein degradation through quantification of the color in chromoprotein expression.
<br><br>
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</p></center>
 
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<br>
 
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<br><br>
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<center><img src="https://static.igem.org/mediawiki/2017/5/5d/T--Lambert_GA--system.png" style="width:70%; margin:auto;"></center>
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<br><br>
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<br><br>
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</p>
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<div class="center">
 
<div class="center">
<img class="img-responsive" style="width:600px" src="https://static.igem.org/mediawiki/2017/c/cc/T--Lambert_GA--SSRApathway.png">
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<img class="img-responsive" style="width:600px" src="https://static.igem.org/mediawiki/2017/b/b2/T--LambertGA--camera2.jpg">
 
</div>
 
</div>
 
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<br>
 +
<center><h3 style="color: #D49AE6;">Development</h3></center>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">One of the most critical variables in imaging today is the consistency of light. Different wavelengths, intensity, power, wattage, and even angling of lights can affect the result of camera imaging. To this end, the ChromQ Light Chamber is designed to control for the variable of light. The box’s dimensions are 12” x 12” x 12” and feature four mini LED-bulbs at 0.6 Watts each with a power source of 3 Volts. Each LED light is positioned 7” down from the top, 45° below the horizontal, one on each side of the chamber, excluding the top and the bottom. The combination of the four lights creates a spotlight that will focus on the specimen being imaged. The top features a circular 2”-diameter cut-out that provides an opening for camera lens to image. The bottom of the chamber is left open in order to properly accommodate and shift any reasonably-sized and –shaped specimen that will be illuminated and imaged with the chamber. The dimensions have been optimized for ideal imaging distance and lighting amount. </p>
 +
<br>
 +
<center><h3 style="color: #D49AE6;">On-Site Application</h3></center>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">Current methods of testing for nutrient deficiency are incredibly time-consuming and require a plethora of steps.  Specimens gathered on site must be sent overseas for testing and the results are returned weeks and even months later. For areas that urgently need immediate care, this lengthy result turn-around time could cost dozens of lives. To solve this pressing issue, the 3D-printed ChromQ Light Chamber is designed to be easily assembled and then taken apart, much like building with Lego pieces. This aspect was created for ease of transportation, as well as ability to be used in the field as part of on-site testing and data collection. Researchers and healthcare providers can simply use the chamber to test for nutrient deficiency at the location of examination and obtain immediate results without having to send specimens to labs overseas. The increase in efficiency revolutionizes the future of point-of-care treatment.  </p>
 +
<br>
 +
<center><h3 style="color: #D49AE6;">Inexpensive</h3></center>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">As a high school lab, one of the many problems we face in research is the lack of funding and monetary support. Sophisticated equipment is incredibly expensive, and so it is very hard to afford the necessary tools and measurement systems for our research. The ChromQ Light Chamber is designed to conquer the issue of the cost of a fluorimeter – instead of visualizing with fluorescence, the project uses  chromoproteins, which can then be imaged with the chamber we built, and results can be obtained that way. This technology can be used by other teams and facilities for their research, and the full plan of the design is available below so that any team can build it themselves.  </p>
 +
<br>
 +
<center><h3 style="color: #D49AE6;">Build Guide</h3></center>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">
 +
<b>Tools</b>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">1. 3D Printer (Build Area of at least 7 x 7 x 8in)
 +
<br>
 +
2. Super Glue
 +
<br>
 +
3. Sanding tools
 +
<br>
 +
4. Non-Reflective Spray Paint(Optional) </p>
 
<br><br>
 
<br><br>
  
 +
<b>Parts</b>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">1. All the STL files included within the file(5 Pieces).
 +
<br>
 +
2. LED Lights(At least 4 LED bulbs)
 +
<br>
 +
3. Camera </p>
 
<br><br>
 
<br><br>
  
 
+
<b>Tips:</b>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">For best results, print at a slow speed to ensure the best outcome possible and make sure the 3D printer is tuned correctly. Print each of the Parts at any desired infill percentage and layer height. Also, it’s recommended to print with brims so it does not curl when printing. If need be, the SLDPRT versions are included in the file. </p>
 
<br><br>
 
<br><br>
  
</p>
+
<b>Instructions</b>
  
<div class="center">
+
<p style="width: 100%; margin: auto; font-size: 16px;">1. Transfer all the STL files into preferred slicing software. <br>
<img class="img-responsive" style="width:300px" src="https://static.igem.org/mediawiki/2017/e/e0/T--Lambert_GA--clpxpanimation.gif">
+
2. Change settings accordingly to the printer. <br>
</div>
+
3. Orient the parts where it will create the least amount of support for best results. <br>
 +
4. Convert it into GCODE files. <br>
 +
5. Transfer the GCODE files into preferred printing software. <br>
 +
6. Start Printing. Print 4 copies of Parts 1, 3, 4, and 5. Print 5 copies of Part 2. <br>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">7. Carefully remove the prints. <br>
 +
8. Sand any defects and remove support materials on the prints. <br>
 +
9. Test that all parts fit correctly. <br>
 +
10. Lightly sand the connecting surfaces for each piece for the best adhesion. <br>
 +
11. Arrange the 4 prints of Part 1 into a square and glue them. <br><br> </p>
  
<br><br>
+
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/4/4e/T--LambertGA--camerainstructions1.png"></center><br><br>
 
+
<p style="width: 100%; margin: auto; font-size: 16px;">12. Insert 4 prints of Part 2 onto its designated location and glue them. </p><br><br>
 
+
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/0/05/T--LambertGA--camerainstructions2.png"></center><br><br>
<br><br><br>
+
<p style="width: 100%; margin: auto; font-size: 16px;">13. Connect Part 5 on top of Part 4 and glue them.</p><br><br>
</p>
+
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/e/e7/T--LambertGA--camerainstructions3.png"></center><br><br>
</div>
+
<p style="width: 100%; margin: auto; font-size: 16px;">14. Repeat Step 13 for 3 more times.<br>
</div>
+
<p style="width: 100%; margin: auto; font-size: 16px;">15. Arrange 2 of the 4 Parts side by side and glue them along with the last Part 2 print.</p><br><br>
</div>
+
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/e/eb/T--LambertGA--camerainstructions4.png"></center><br><br>
</div>
+
<p style="width: 100%; margin: auto; font-size: 16px;">16. Repeat the rest as mentioned in Step 15 and glue them to the Part created in Step 15. </p><br><br>
</section>
+
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/6/61/T--LambertGA--camerainstructions5.png"></center><br><br>
 
+
<p style="width: 100%; margin: auto; font-size: 16px;">17. Place the Assembly created in Step 16 onto the Assembly created in Step 12 and glue them all together. </p> <br><br>
<h2> Improving Previous Parts </h2>
+
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/d/d1/T--LambertGA--camerainstructions6.png"></center><br><br>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">18. Insert the 4 prints of Part 3 into the 4 holes of the side of the box and glue them. </p><br><br>
 +
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/d/d0/T--LambertGA--camerainstructions7.png"></center><br><br>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">19. Insert the LED lights into the holes present in the Part 3 prints. (LED lights: 0.6 Watts, 20mA current; solder into series circuit with 2 9-volt batteries) </p>
 +
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/a/a8/T--LambertGA--realcamera1.jpg"></center><br><br>
 +
<p style="width: 100%; margin: auto; font-size: 16px;">20. If needed, spray some non-reflective paint inside the box to seal and block out light, for best results spray several coats. <br></p> <br>
 +
<center><img class="img-responsive" style="width:500px" src="https://static.igem.org/mediawiki/2017/2/27/T--LambertGA--lightbox.png"></center><br><br>
 +
<b>*Note: If you are interested in 3D-printing the ChromQ Light Chamber, please contact us at igem.lamb@gmail.com for the program print files. </b> </p>
 
<br>
 
<br>
<div class="column full_size" style="width:70%; margin:auto;" >
+
<center> <h2>  References </h2> </center>
<div>
+
<p>Dhakar, L. (n.d.). Image Color Picker (Z. A., Ed.). Retrieved October 10, 2017, from
 
+
http://www.colorcodepicker.com/
 
<br><br>
 
<br><br>
 
+
Purple color codes. (n.d.). Retrieved October 10, 2017, from
 +
http://www.rapidtables.com/web/color/purple-color.htm
 
<br><br>
 
<br><br>
 
+
RGB Color Gradient Maker. (n.d.). Retrieved October 10, 2017, from http://www.perbang.dk/rgbgradient/
</div>
+
 
<br><br>
 
<br><br>
 +
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi, S., . . . Hara-Nishimura, I. (2003,
 +
September). Why green fluorescent fusion proteins have not been observed in the vacuoles of
 +
higher plants. The Plant Journal, 35(4), 545-555. doi:10.1046/j.1365-313X.2003.01822.x
 +
</p>
 +
  
<h2>  References </h2>
 
<p style="color: white">
 
And, S. A. (2009, February 13). Sarita Ahlawat. ClpXP Degrades SsrA-tagged proteins in S.pneumoniae.Retrieved Summer, 2017, from http://jb.asm.org/content/191/8/2894.full
 
<br><br>
 
Andersen , J.B. , Sternberg , C. , Poulsen , L.K. , Bjorn , S.P. , Givskov , M. , and Molin , S. ( 1998 ) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria . Appl Environ Microbiol 64 : 2240 – 2246 .
 
<br><br>
 
Baker, T. A., & Sauer, R. T. (2011, June 27). ClpXP, an ATP-powered unfolding and protein-degradation machine. Retrieved Summer, 2017, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209554/
 
<br><br>
 
Bar-Nun, S., & Glickman, M. H. (2012). Proteasomal AAA-ATPases: Structure and function. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1823(1), 67–82. doi:10.1016/j.bbamcr.2011.07.009. Retrieved Summer, 2017 from http://www.sciencedirect.com/science/article/pii/S0167488911001984
 
<br><br>
 
Bohn , C. , Binet , E. , and Bouloc , P. ( 2002 ) Screening for stabilization of proteins with a trans-translation signature in Escherichia coli selects for inactivation of the ClpXP protease . Mol Genet Genomics 266 : 827 –831 .
 
<br><br>
 
Burton , R.E. , Siddiqui , S.M. , Kim , Y.I. , Baker , T.A. , and Sauer , R.T. ( 2001 ) Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine . EMBO J 20 : 3092 –3100 .
 
<br><br>
 
Ciechanover, A. (2005). Cell death and differentiation - abstract of article: Intracellular protein degradation: From a vague idea thru the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting[ast]. Cell Death & Differentiation, 12(9), 1178–1190. doi:10.1038/sj.cdd.4401692
 
<br><br>
 
Cooper, G. M. (2000). Protein degradation. Retrieved Summer, 2017 from http://www.ncbi.nlm.nih.gov/books/NBK9957/
 
<br><br>
 
Farrell, C., Grossman, A., & Sauer, R. (2005). Cytoplasmic degradation of ssrA-tagged proteins.Molecular microbiology., 57(6), 1750–61. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16135238
 
<br><br>
 
Flynn , J.M. , Levchenko , I. , Seidel , M. , Wickner , S.H. , Sauer , R.T. , and Baker , T.A. ( 2001 ) Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis . Proc Natl Acad Sci USA 11 : 10584 – 10589.
 
<br><br>
 
Georgia Institute of Technology. (2015, September 1). “Bacterial litmus Test” provides inexpensive measurement of Micronutrients. Retrieved from GT News Center, http://www.news.gatech.edu/2015/09/01/bacterial-litmus-test-provides-inexpensive-measurement-micronutrients
 
<br><br>
 
Goldberg, A.L., A.S. Menon, S. Goff and D.T. Chin. 1987. The mechanism and regulation of the ATP-dependent protease La from Escherichia coli. Biochem. Soc. Trans. 15: 809-811. Retrieved October 1, 2017 from http://www.fao.org/wairdocs/ilri/x5550e/x5550e0d.htm
 
<br><br>
 
Hwang BJ, Woo KM, Goldberg AL, Chung CH. Protease Ti, a new ATP-dependent protease in Escherichia coli,contains protein-activated ATPase and proteolytic functions in distinct subunits. J Biol Chem. 1988;263:8727–8734.
 
<br><br>
 
Katayama-Fujimura Y, Gottesman S, Maurizi MR. A multiple-component, ATP-dependent protease from Escherichia coli. J Biol Chem. 1987;262:4477–4485.
 
<br><br>
 
Landry, B. P., & Stöckel, J. (2013). Use of degradation tags to control protein levels in the Cyanobacterium Synechocystis sp. Strain PCC 6803. Applied and Environmental Microbiology,79(8), 2833–2835. doi:10.1128/AEM.03741-12
 
<br><br>
 
Lee C, Schwartz MP, Prakash S, Iwakura M, Matouschek A. ATP-Dependent Proteases Degrade Their Substrates by Processively Unraveling Them from the Degradation Signal.
 
<br><br>
 
McNerney, M. P., Watstein, D. M., & Styczynski, M. P. (2015). Precision metabolic engineering: The design of responsive, selective, and controllable metabolic systems. Metabolic Engineering, 31, 123–131. doi:10.1016/j.ymben.2015.06.011
 
<br><br>
 
Minikel, E. V. (2013, June 11). Basics of protein degradation. Retrieved Summer, 2017, from http://www.cureffi.org/2013/07/11/basics-of-protein-degradation/
 
<br><br>
 
Mogk A, Schmidt R, Bukau B. The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies. Trends Cell Biol. 2007;17:165–172.
 
<br><br>
 
Purcell, O., Grierson, C. S., Bernardo, M. di, & Savery, N. J. (2012). Temperature dependence of ssrA-tag mediated protein degradation. Journal of Biological Engineering, 6(1), . doi:10.1186/1754-1611-6-10
 
<br><br>
 
Schrader, E. K., Harstad, K. G., & Matouschek, A. (n.d.). Targeting proteins for degradation. , 5(11), . Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228941/
 
<br><br>
 
Snider, J., Thibault, G., & Houry, W. A. (2008). The AAA+ superfamily of functionally diverse proteins. , 9(4), . Retrieved Summer, 2017 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643927/
 
<br><br>
 
Tanaka K. The proteasome: overview of structure and functions. Proc Jpn Acad Ser B Phys Biol Sci.2009;85:12–36.
 
<br><br>
 
Tao, L., & Biswas, I. (2015). Degradation of SsrA-tagged proteins in streptococci. , 161(Pt 4),. Retrieved September 9, 2017 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857447/
 
<br><br>
 
Tu, D., Lee, J., Ozdere, T., Lee, T. J., & You, L. (2007, January ). Engineering Genetic Circuits: Foundations and Applications. Retrieved from http://people.duke.edu/~you/publications/Tu_etal_SyntheticBiology.pdf
 
<br><br>
 
Watstein, D. M., McNerney, M. P., & Styczynski, M. P. (2015). Precise metabolic engineering of carotenoid biosynthesis in Escherichia coli towards a low-cost biosensor. Metabolic Engineering,31, 171–180. doi:10.1016/j.ymben.2015.06.007
 
<br>
 
</p>
 
 
<div class="button_click"  onClick=" parent.location= 'https://2017.igem.org/Special:Upload '">
 
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<a href="http://www.forsyth.k12.ga.us/lhs"><img src="https://static.igem.org/mediawiki/2017/9/9e/T--LambertGA--longhorn_iGEM_logo.png" class="transparent" style="height:100px;padding:20px;"></a>
  
 
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Revision as of 11:32, 25 October 2017


Hardware



Function

The ChromQ Light Chamber is a 3D-printed imaging measurement system used to quantify results of nutrient deficiency. For our current project, it is also used to quickly and inexpensively measure relative protein degradation through quantification of the color in chromoprotein expression.



Development

One of the most critical variables in imaging today is the consistency of light. Different wavelengths, intensity, power, wattage, and even angling of lights can affect the result of camera imaging. To this end, the ChromQ Light Chamber is designed to control for the variable of light. The box’s dimensions are 12” x 12” x 12” and feature four mini LED-bulbs at 0.6 Watts each with a power source of 3 Volts. Each LED light is positioned 7” down from the top, 45° below the horizontal, one on each side of the chamber, excluding the top and the bottom. The combination of the four lights creates a spotlight that will focus on the specimen being imaged. The top features a circular 2”-diameter cut-out that provides an opening for camera lens to image. The bottom of the chamber is left open in order to properly accommodate and shift any reasonably-sized and –shaped specimen that will be illuminated and imaged with the chamber. The dimensions have been optimized for ideal imaging distance and lighting amount.


On-Site Application

Current methods of testing for nutrient deficiency are incredibly time-consuming and require a plethora of steps. Specimens gathered on site must be sent overseas for testing and the results are returned weeks and even months later. For areas that urgently need immediate care, this lengthy result turn-around time could cost dozens of lives. To solve this pressing issue, the 3D-printed ChromQ Light Chamber is designed to be easily assembled and then taken apart, much like building with Lego pieces. This aspect was created for ease of transportation, as well as ability to be used in the field as part of on-site testing and data collection. Researchers and healthcare providers can simply use the chamber to test for nutrient deficiency at the location of examination and obtain immediate results without having to send specimens to labs overseas. The increase in efficiency revolutionizes the future of point-of-care treatment.


Inexpensive

As a high school lab, one of the many problems we face in research is the lack of funding and monetary support. Sophisticated equipment is incredibly expensive, and so it is very hard to afford the necessary tools and measurement systems for our research. The ChromQ Light Chamber is designed to conquer the issue of the cost of a fluorimeter – instead of visualizing with fluorescence, the project uses chromoproteins, which can then be imaged with the chamber we built, and results can be obtained that way. This technology can be used by other teams and facilities for their research, and the full plan of the design is available below so that any team can build it themselves.


Build Guide

Tools

1. 3D Printer (Build Area of at least 7 x 7 x 8in)
2. Super Glue
3. Sanding tools
4. Non-Reflective Spray Paint(Optional)



Parts

1. All the STL files included within the file(5 Pieces).
2. LED Lights(At least 4 LED bulbs)
3. Camera



Tips:

For best results, print at a slow speed to ensure the best outcome possible and make sure the 3D printer is tuned correctly. Print each of the Parts at any desired infill percentage and layer height. Also, it’s recommended to print with brims so it does not curl when printing. If need be, the SLDPRT versions are included in the file.



Instructions

1. Transfer all the STL files into preferred slicing software.
2. Change settings accordingly to the printer.
3. Orient the parts where it will create the least amount of support for best results.
4. Convert it into GCODE files.
5. Transfer the GCODE files into preferred printing software.
6. Start Printing. Print 4 copies of Parts 1, 3, 4, and 5. Print 5 copies of Part 2.

7. Carefully remove the prints.
8. Sand any defects and remove support materials on the prints.
9. Test that all parts fit correctly.
10. Lightly sand the connecting surfaces for each piece for the best adhesion.
11. Arrange the 4 prints of Part 1 into a square and glue them.



12. Insert 4 prints of Part 2 onto its designated location and glue them.





13. Connect Part 5 on top of Part 4 and glue them.





14. Repeat Step 13 for 3 more times.

15. Arrange 2 of the 4 Parts side by side and glue them along with the last Part 2 print.





16. Repeat the rest as mentioned in Step 15 and glue them to the Part created in Step 15.





17. Place the Assembly created in Step 16 onto the Assembly created in Step 12 and glue them all together.





18. Insert the 4 prints of Part 3 into the 4 holes of the side of the box and glue them.





19. Insert the LED lights into the holes present in the Part 3 prints. (LED lights: 0.6 Watts, 20mA current; solder into series circuit with 2 9-volt batteries)



20. If needed, spray some non-reflective paint inside the box to seal and block out light, for best results spray several coats.




*Note: If you are interested in 3D-printing the ChromQ Light Chamber, please contact us at igem.lamb@gmail.com for the program print files.


References

Dhakar, L. (n.d.). Image Color Picker (Z. A., Ed.). Retrieved October 10, 2017, from http://www.colorcodepicker.com/

Purple color codes. (n.d.). Retrieved October 10, 2017, from http://www.rapidtables.com/web/color/purple-color.htm

RGB Color Gradient Maker. (n.d.). Retrieved October 10, 2017, from http://www.perbang.dk/rgbgradient/

Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi, S., . . . Hara-Nishimura, I. (2003, September). Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. The Plant Journal, 35(4), 545-555. doi:10.1046/j.1365-313X.2003.01822.x