1.Documentation of Part: BBa_J04500

(IPTG inducible promoter with RBS)

J04500 is a composite part containing R0010 and B0034. It is a LacI regulated promoter with RBS. This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first site can bind with CAP protein, which is generally present in E. coli and is associated with cell health and availability of glucose. Binding with CAP can promote the expression of its downstream genes. The second site binds with LacI protein, which negatively regulate the downstream genes. LacI is the coding repressor protein in E. coli. This repression of LacI can be removed by IPTG, which means the IPTG disable the binding of LacI to the promoter, so as to induce the expression of downstream genes.

In order to test the inhibition efficiency of LacI and the induction of IPTG to this part, we connected E0040 and B0015 parts with the J04500 together. Then we test the GFP expression with and without IPTG presence. The data are showed in Fig.1 and Fig.2.

Fig.1 the GFP expression with and without IPTG (0.5mM) presence in E. coli.

Fig.2 the GFP expression with and without IPTG (2mM) presence in E. coli.

As the data suggest, this promoter is an IPTG inducible promoter in E. coli. There is distinct difference in signals with or without IPTG induction. As expected, under IPTG induction, there is about 3-fold increase in GFP signals compared to those without IPTG presence. However, we can still find the leaking expression, compared with the negative control in E. coli.

This part is incompatible with species containing active LacI coding regions. However, our experiment showed that it can initiate the transcription of downstream genes in E. coli even if there is no IPTG presence, possibly because that the LacI expressed in the cells is not sufficient for repression this promoter. GFP have some leakage expression without the presence of IPTG.

2.Printing Instructions of 3D Straw for Our Project:

1)Software: PrusaSlicer 2.2.0

2)Adjustment Procedures:

a)Search Google for “Thingiverse”, open link in browser.

b)In the search bar, enter “straw”, and choose this pre-designed 3-D model. It contains multiple files of multiple shapes of straw.

c)Click “Download Files”. Wait for it to download.

d)Find the location of download, right click on the zipped file, and click on “Extract all”.

e)Open software “PrusaSlicer 2.2.0”. Go to the top bar and click on “File > Import > Import STL/OBJ/ AMF...”

f)Select and open the file just downloaded.

g)In this given file, there are multiple forms of straw to be printed, and we chose the following 4 of them.

h)To adjust its size, click on the model you want to adjust, and select “Scale (S)” on the left function bar. As demonstrated in the screenshot, the width, length and height of the object is the distance between the 2 corresponding red, orange, and blue square cubes. Drag to make changes to the size of the object.

i)How to change directions of an object: select the object, and click on “Rotate”. Be sure to choose the z-axis that spins the object without having it deviating from the printer bed.

j)When finished adjusting and the objects are ready to print, go to the right column and select the appropriate printer to use. Each style of printer is suited for only one type of G-code.

k)Choose a support for the object. The machine cannot print in mid-air; choose “support on build-plate only” for this specific print.

l)Choose infill. This is the density of the object to be printed. Remain its default “15%” for this specific print.

m)Go to the right bottom of the interface, and click “Slice”. This icon will plan the layers the printer will be executing.

n)Insert the SD card from the 3D printer, then click “export G-code” to it. Save the file in its default format, then safely eject the SD card.

3)Printing

a)From the printer, unload the filament from the previous print.

b)Choose PLA material. Wait for the filament to be heated until its liquid, then quickly pull out the previous filament.

c)Choose a new filament to print with. We Wechoose the semi-transparent color. Load the new filament onto the printer.

d) The printer will secrete a mixed coloration of the old and new filament color. Choose “no” if the color is not your selected color. Only answer yes to the question unless the old filament color is gone. Dispose the secreted material.

e)Insert SD card into the printer. Select the desired file, press the knob to “start”.

f)The printer first heats the bed, then measures it to make sure the dimensions in the G-code fit onto the bed’s printable areas on the metal bed.