Summary

Our bronze medal contribution comprises of three projects: we have used CAD to 3D print a shaking incubator rack and documented instructions on how to do so, we have characterised 3 JUMP (Joint Universal Modular Plasmid) Destination Vectors, of varying copy number, for future synthetic biologists to reference, and we have also provided instructions on how iGEM teams can create DIY Team T-shirts. We believe these contributions are meaningful and exciting, and are described in detail below.

1. Print Your Own Shaking Incubator Rack

Project Motivation:

• Liquid culture is incubated at 37^oC in an incubator in order for bacteria cells to grow after inoculation. The incubator is set to shaking as it increases the oxygen penetration into the media and tubes can also be set at an incline so that the surface area of the media in contact with the air is greater, and shaking is more efficient.
• As we only had vertical tube racks in our lab, we needed to source one.
• On the market, the prices range from $200-$500 with even a support to put an existing rack at an incline being about $30. So, we needed to find a cheaper option. We have access to two 3D printers in the Jim Haseloff lab, Department of Plant Sciences so we decided to print our own! This way we could customise the rack, with it fitting our incubator perfectly whilst also keeping our costs low. We know that funding is often an obstacle for many iGEM teams and even for many start-ups so we thought we could share our CAD (computer aided design) publicly to inspire others to create their own financially-accessible lab equipment.
• For this 3D-print, it took me a few days to get used to the software as I hadn’t used CAD since secondary school but eventually I got a hang of the tools. I was using a picture reference of a previously printed rack. I did a few trials with just attempting to make the silhouette of the rack before being suggested that i make the cross-section and then extrude that so it prints as one piece and I don’t need to assemble anything afterwards unlike in the reference photo.
• See below the detailed instructions for how to make your own 3D-printed shaking incubator rack, or download the instruction manual in pdf format here: Instruction Manual



How to Make Your Own 3D Printed 'Shaking Incubator Rack': Instructions




Following these instructions, I estimate it takes a few hours to design the rack and get the print going then the print itself will take 1 day and 20 hours.
Required materials:
3D Printer (Ultimaker S3 or S5, depending on size of print - both would have fitted this rack but I used S5)
Ultimaker PLA - Black (but any colour could be used)
You will need to download:
Autodesk Fusion 360 (for the CAD)
Ultimaker Cura (if you have an Ultimaker 3D printer, specifically for the printing process)






Step 1: Construct the Cross Section:

1. Download Autodesk Fusion 360: https://www.autodesk.co.uk/campaigns/education/fusion-360
2. Create ‘New sketch’
3. Choose shape ‘centre rectangle’ and make a 49.497mm x 49.497mm square.
4. Delete the top and right hand side of the square, and turn the hypotenuse construction line into a material line to make the right angle triangle.
5. Finding the mid point and the two 45 degree vertices, make a perpendicular line of 32mm outwards.













Step 2: Extrude the Cross Section:

1. Using the hollow extrude button, extrude the cross section 240mm at 5mm thickness.
2. In our case, the middle rack didnt extrude so we used the thicken tool to thicken it 2.5mm either side. That left behind the extra mid line and joining line (Fig 2.1). We will get rid of these in the following steps.
3. Using the combine tool, remove the line from the middle rack as we want this to print in one piece.



Step 3: Add Holes:

1. Create ‘New Sketch’ and select the top rack rectangle as the plane.
2. Use the Create > point tool to find the midpoint of the width of the rectangle and make 8 points for the centres of the holes.
3. Use the dimension tool to make sure you follow the distances in the image. There should be 15mm from the edge to the centre of the edge circles and 30mm between the rest of the circle’s centres (see Fig.3.1).
4. Create holes on all 8 points, following these measurements. The holes will go straight through the 2 racks and slightly through the 3rd, leaving a well for the tubes to rest at the bottom in and be less likely to fall about (see Fig.3.2).
5. Here you will notice the issue with the mid line in the middle rack which prevents you seeing right through all the holes. You can now select this plane and delete it (see Fig.3.3.).
















Step 4: Add Sides:

1. Sketch the cross sectional area of the side and extrude outward for 5mm. Make sure this remains as one piece with the rest of the structure and do the same for the other side.












Step 5: Text (Optional Personalisation):

1. We’ve inscribed ‘iGEM 2022’ on the back of the rack, to make it stand out against the other racks in our communal lab but this can be altered to your preference.
2. To personalise, create ‘New sketch’ and select the ‘text’ option.
3. Once you’ve typed it, finish sketch.
4. Extrude this -1mm into the plane.




Step 6: Add a Base:

Adding a base will help to improve the stability of the rack.


1. Creating a new sketch with a centre rectangle on the plane of the base of the structure, make a rectangle with dimensions 286mm x 89.497 (creating a 20mm brim around the base- see Fig.6.1). This can be made bigger if things are needing to be added to the base.








Step 7: Fillet:

In order to make the final product look smoother and less pointy, we will carry out a series of filleting and chamfering different edges.

1. First we fillet the joint between the structure and the base, with diameter 2mm, by highlighting the 4 edges around that join and using the fillet tool (Fig.7.1). Having a smoother transition between parts also helps increase the quality and strength of the final print.
2. Next select the outer edges and use the filleting tool to round them all off (Fig.7.2).





Step 8: Upload to Ultimaker Cura:

1. Download Ultimaker Cura. We are using version 5.1.0. See Fig.8.1 and Fig.8.2 to see settings used.
2. We are using the Ultimaker S5 to print, you will need to select your 3D printer.
3. Select your printing material - we are using Strong black PLA (listed as ‘PLA’ on the machine for us- see Fig.8.4). Make sure that you’ve added supports to the print.
4. Press ‘slice’ to see how it will be printed and how long it will take (Fig.8.5 and Fig.8.6).
5. Use the right hand side toggle to expose the layers that will be printed. The supports are shown in turquoise in Fig.8.8.
6. You can use the bottom toggle to play through the printer head, printing the rack.
7. Remove the USB from the ultimaker printer and insert into the laptop.
8. Save the Ultimaker Cura file ‘to removable drive’
9. If the printer finds any issues, it will flag them here. If not, press print! The waiting game begins...








































Fig.8.10.

Step 9: Remove Rack:

1. Once the print is finished, gently prise it from the printer bed.
2. Snap off the brim from the edge of the base.
3. Snap off the support structures, the have lower infill density than the print so should be relatively easy to.
4. (Optional) You can sand down the final rough edges







Step 10: Shake Your Test Tubes!

You can now use your new test tube rack!







2. JUMP DV Characterisation

Contents:
1. How are we contributing?
2. How did we do it?
3. Wet-lab Diary
4. Results



1. How are we contributing?

For the contribution criteria we characterised 3 of the JUMP (Joint Universal Modular Plasmid) destination vectors, of varying copy number, found in this year’s distribution kit.
In summary, we found that if you are expressing a protein in a low copy number plasmid you can add up to 540mM of ammonium sulphate, up to 360mM if you are working in low-med copy number plasmid and 0mM to a high copy number plasmid for greater expression.

With JUMP cloning gaining popularity, having a better understanding about the effect of plasmid copy number and external stresses on these plasmids will give future iGEM teams and synthetic biologists a better idea of which copy number plasmid would be best for their cloning application.
During one of our Integrated Human Practice discussions with protein engineering company, Codexis, we discussed common perturbations that cells are likely to be subjected to in industry, and we were informed about ammonium build up in bioreactors in the case of insufficient mixing.

After reading ‘Ammonium Toxicity in Bacteria’ by Müller et al., 2005, it was decided that testing ammonium sulphate concentrations from 0-1000mM was a reasonable range in order to gather data within and beyond the scope of their testing parameters. The paper claims that ‘in E. coli, addition of 750 mM and 1000 mM ammonium (375 mM and 500 mM (NH4)2SO4) impaired growth’ and with 0-1000mM ammonium sulphate, we aimed to record all stages of detriment to the point at which the bacteria are no longer able to cope.

Our contribution is also recorded on the Part:BBa_J428351 (pSC101 low-copy no. JUMP DV) Registry page - this includes our most important contributory data summarised on the main page:

Part:BBa_J428351 Registry page



2. How did we do it?

To carry out the experiment we used a Clariostar Plate Reader (with permission from Jim Haseloff’s lab - Plant Sciences, Cambridge University) and 96 well plates to record the growth (OD600) and sfGFP fluorescence intensity of JUMP destination vectors: pSC101 low copy no. pJUMP27-1A, pBBRI low-med copy no. pJUMP23-1A and pUC high copy no. pJUMP28-1A in Neinhardt media of varying ammonium concentrations. Along with these, we’ve used 2 negative controls: just EZRDM (Neinhardt media) containing the ammonium concentrations and ‘just cells’ being DH5α E. coli with ammonium too. Our first JUMP level 1 plasmid acted as our positive control, expressing sfGFP. Within the limited number of wells, we were able to do 3 technical replicates for each condition.

See below for the plate layout and Wet-lab protocols:





3. Wet-Lab Diary:

Firstly, we’d like to thank:


• Haseloff lab for Plate reader use and space to carry out this characterisation
• Our Manager Camillo Moschner for overseeing this experiment at Plant Sciences
• Instructor Ignacy Bonter for granting us access to the Plant Sciences lab
• Instructors Georgeos Hardo and Charlie Wedd for giving us access to ammonium sulphate


31/08/22: Preparations


1. Transform 4E, 2M and 4I from DK Plate 1:
        4E = pSC101 low copy no. pJUMP27-1A      2M = pBBRI low-med copy no. pJUMP23-1A      4I = pUC high copy no. pJUMP28-1A
   
2. Tranform any controls too

For transformation protocol, see: 'Wetlab > Protocols > Transformation'

05/09/22 - Innoculating liquid Culture - test 1


1. We want 500mL EZRDM so so weigh out 16.45g Neinhardt basal salt mixture (room temp.), 1.55g Neinhardt supplement mixture to a 50mL Falkon Tube (fridge)
   
2. Vacuum sterilise EZRDM before use
   
3. Label 5 snap caps with plasmid/sample name: ‘Just cells’ (neg. control), ‘pSC101’ (low copy no.), ‘pBBR1’ (low-med. copy no.), ‘pUC’ (high copy no.), ‘lvl1’ (neg. Control, no sfgfp).
   
4. 2mL of EZRDM added to all 5 with stripette
   
5. In all but ‘just cells’, 2uL of Kan antibiotic is added.
   
6. Pick colonies and innoculate liquid culture
   
7. Incubate for 24 hours at 30 degrees (at 11:30am) at 180rpm
   
   
06/09/22 - Well Plate - test 1





1. Add 20mL EZRDM to ammonium sulphate powder
   
2. Vortex until dissolved
   
3. Syringe filter with [Millipore Express PES Membrane Filter unit, 0.22um]
4. Label 5 snapcaps with the 5 ammonium sulphate concentrations:
       -1000mM: 4mL 1000mM ([NH4]2SO4)
       -750mM: 3mL 1000mM ([NH4]2SO4) & 1mL 0mM (EZRDM)
       -500mM: 2mL 1000mM ([NH4]2SO4) & 2mL 0mM (EZRDM)
       -250mM: 1mL 1000mM ([NH4]2SO4) & 3mL 0mM (EZRDM)
       -0mM: 750mM: 4mL 0mM (EZRDM)
   
5. Pipette up 10mL of [NH4]2SO4 using stripette and release 4ml,3ml,2ml,1ml into 1000mM,750mM,500mM,250mM. (leave 0mM empty)
   
6. Then pipette up 10mL of EZRDM using stripette and release 4ml,3ml,2ml,1ml into 0mM, 250mM, 500mM, 750mM. (leave 1000mM alone)
     -They should all have final vol. of 4mL
   
7. ‘Just cells’ will be the only wells with things in without Kanamycin. So pipette 3x 200uL of each stock (into the wells with their replicates: ‘Just cells’ = 0,0,0,250,250,250,500,500,500,750,750,750,1000,1000,1000.
     -All the stock now need Kanamycin added so 600uL is gone from each, leaving 3400uL left.
   
8. Add 3.4uL Kan to all the 5 stocks - our just EZRDM with diff ammonium sulphate conc.s wells will also have Kanamycin
   
9. Pipette 200uL of the respective stocks into their wells
   
10. Pipette 1uL of the respective cells into their wells.
    
11. Put transparent film on top of well plate and put in plate reader.
    
12. Each cycle should take less than 4mins 30s
    
    
07/09/22 - Liquid Culture no.2


1. Make more liquid culture for the next rerun
   
2. Label 5 snap caps with plasmid/sample name: ‘just cells’, ‘lvl 1’, ‘pSC101’, ‘pBBR1’, ‘pUC’
   
3. 2mL of EZRDM added to all 5 with stripette - stripette up 10mL EZRDM and release 2ml into each
   
4. In all but ‘just cells’, 2uL of Kan antibiotic is added
   
5. Pick colonies and innoculate liwuid culture
   
6. Incubate for 24 hours at 30 degrees (at 11:30am) at 180rpm
   
  -Looking at preliminary data from the plate reader, we can narrow down our scope to no more than 750mM for our ammonium sulphate concentration
  -Could do 750mM and below but that leaves us with decimals - 720mM works nicely.

08/09/22- Well Plate Pipetting no.2








1. Add 20mL EZRDM to ammonium sulphate powder
   
2. Vortex until dissolved
   
3. Steralise with syringe filter [Millipore Express PES Membrane Filter unit, 0.22um]
4. Make 720mM stock:
       -14.4mL of 1000mM [NH4]2SO4 & 5.6mL EZRDM (0mM) =20mL
   
5. Label 5 snapcaps with the 5 UPDATED ammonium sulphate concentrations:
       -720mM: 4mL 720mM ([NH4]2SO4)
       -540mM: 3mL 720mM ([NH4]2SO4) & 1mL 0mM (EZRDM)
       -360mM: 2mL 720mM ([NH4]2SO4) & 2mL 0mM (EZRDM)
       -180mM: 1mL 720mM ([NH4]2SO4) & 3mL 0mM (EZRDM)
       -0mM: 750mM: 4mL 0mM (EZRDM)
   
6. Pipette up 10mL of [NH4]2SO4 using stripette and release 4ml,3ml,2ml,1ml into 720mM, 540mM, 360mM, 180mM. (leave 0mM empty)
   
7. Then pipette up 10mL of EZRDM using stripette and release 4ml,3ml,2ml,1ml into 0mM, 180mM, 360mM, 540mM. (leave 720mM alone)
     -They should all have final vol. of 4mL
   
8. ‘Just cells’ will be the only wells with things in without Kanamycin. So pipette 3x 200uL of each stock (into the wells with their replicates: ‘Just cells’ = 0,0,0,180,180,180,360,360,360,540,540,540,720,720,720.
   
9. All the stock now need Kanamycin added so 600uL is gone from each, leaving 3400uL left.
   
10. Add 3.4uL Kan to all the 5 stocks - our just EZRDM with diff ammonium sulphate conc.s wells will also have Kanamycin
    
11. Pipette 200uL of the respective stocks into their wells
    
12. Pipette 1uL of the respective cells into their wells.
    
13. Put transparent film on top of well plate and put in plate reader.
    
    

4. Results


Most interesting results: 3 JUMP DV’s Expression with increasing [NH4]2SO4 concentration:



For all copy number plasmids, ammonium sulphate of any concentration is detrimental to growth however:

pSC101 (low copy no.): Ammonium enhanced fluorescence intensity up to 540mM. Despite the fluorescence rate decrease, the maximum fluorescence increased as we increased ammonium sulphate concentration to 540mM with the cells reaching their limit at 720mM. Therefore, up to a maximum of 540mM of ammonium sulphate can be added to the growth media of cells containing pSC101 DV in order to enhance the expression of the protein of interest (in place of our sfGFP).

pBBR1 (low-med copy no.): Ammonium enhances fluorescence intensity up to 360mM. Despite the fluorescence rate decrease, the maximum fluorescence increases as we increase ammonium sulphate concentration to 360mM with the cells having lower expression at 540mM, reaching their limit at 720mM. Therefore, up to a maximum of 360mM of ammonium sulphate can be added to the growth media of cells containing pBBR1 DV in order to enhance the expression of the protein of interest (in place of our sfGFP).

pUC (high copy no.): For high copy number plasmids, we can report that any ammonium concentration is detrimental to the cells expression. The greatest fluorescence was recorded with 0mM of ammonium sulphate, with decreasing expression as it was increased.

Test 1- 06/09/22
Well Plate Decryption:



Growth Curves: OVERVIEW: 3 JUMP DV’s Growth varying with [NH4]2SO4 concentration :



JUMP DV’s & control's Growth per [NH4]2SO4 concentration :




• At 0mM, pUC, the highest copy number plasmid containing cells has the longest stationary phase and has the greatest amount of growth. This is followed by pBBR1 at med/low copy number, with a similar high growth but dies off quickly. pSC101 is the lowest copy number plasmid and has even less growth. These are the controls to compare the ammonium effects to.
• {From the paper, we were told to expect ammonium “impairs growth” as a “result of general osmotic or ionic effects.”}
• At 250mM [NH4]2SO4, the higher the copy number of the plasmids, the more their growth is affected by the ammonium. pUC is effected the most with the lowest growth, then pBBR1 and pSC101. The metabolic burden of having a higher copy number may be making the E. coli more susceptible to ionic/osmotic effects.
• At 500mM [NH4]2SO4 similar results are observed, with pSC1010 and pBBR1 (lowest) copy number plasmids being effected similarly to each other. BUT more interestingly is that at this level of ammonium, all the cells that contain plasmids have extended growth times, only reaching their maximum growth at the end of the experiment. So, ammonium exposure of a high enough level can slow down the growth rate of the cells regardless their copy number but will ALSO effect the maximum growth reached by higher copy numbers too.
• At 750mM and 1000mM [NH4]2SO4, there is minimal growth - all cells are unable to grow significantly under this high molarity of ammonium.


ANOVAs - Are we seeing significant differences between different copy number plasmids?

• Yes!!
• With our ANOVA plots, the peaks above our dotted line show significant difference between the growth of the plasmids at these times. They were coded with rolling averages in 5 min increments up to 50mins.
• For 0mM:


We see 3 main peaks at stages of growth: exponential, stationary and death
• For 250mM and 500mM:




We only see one peak around the exponential growth phase of the plasmids, signifying that the ammonium has the greatest effect on the growth stage of the bacteria
• For 750mM and 1000mM:




There are no significance peaks here so all the cells are equally detrimentally affected by the high ammonium concentration.

Sigmoidal Curve Fitting & Growth Rate Stats

• Using the sigmoidal function: L / (1 + np.exp(-k*(x-x0))) + b, we fit each of our growth curves to a sigmoidal curve to smoothen them out. As we have quite dramatic death phases, we gave the curve the max values of our data to stop fitting at.
  
• For 0mM:
  
  
  
  
  As copy number increases:
      Midpoint increases (it takes longer for the cells to reach half of their maximum growth - copy number burden delays growth).
      Carrying capacity increases (cells reach higher max. growth)
      dt (doubling time) increases (it takes longer for the cells to double - again, copy number burden)
  
• For 250mM:
  
  
  
  
  With ammonium present, the lowest copy number plasmid, pSC101, has the greatest midpoint (takes the longest to reach half of its maximum growth) - though the rather large carrying capacity is suspicious.
• For 500mM:
  
  
  
  
  Here, the low/med copy number plasmid pBBR1 has the greatest doubling time (slowest growth) and carrying capacity (overall had most growth)
• For 750mM and 1000mM:
  No curves to fit

Fluorescence Intensity Curves:
Here, the plasmids in the distribution kit contain sfGFP which is easy for us to measure. However, our contribution also has a greater significance as this sfGFP can stand in for any coding sequence that can be cloned into these destination vectors. For certain levels of expression, synthetic biologists can use our data to determine which copy number would suit their application best, and how osmotic/ionic effects are portrayed through the expression.

OVERVIEW: 3 JUMP DV’s Fluorescence Intensity varying with [NH4]2SO4 concentration:


JUMP DV’s & controls Fluorescence Intensity per [NH4]2SO4 concentration:



• At 0mM, pUC, the highest copy number plasmid containing cells has a significantly greater expression of sfGFP. This is to be expected as more plasmids = more sfGFP = greater fluorescence intensity {From the paper, we were told to expect ammonium “impairs growth” as a “result of general osmotic or ionic effects.” so let’s investigate its effect on fluorescence} At 250mM [NH4]2SO4, the higher the copy number plasmid, pUC fluorescence has been lowered slightly, but with minimal effect on the lower copy plasmids. The metabolic burden of having a higher copy number may be making the E. coli more susceptible to the ionic/osmotic effects. At 500mM [NH4]2SO4 the pUC fluorescence is lowered significantly, reaching about half of the intensity as half as much ammonium (inverse proportionality?) At 750mM and 1000mM [NH4]2SO4, no fluorescence is observed.


ANOVAs - Are we seeing significant differences in fluorescence between different copy number plasmids?

• Yes!!
• With our ANOVA plots, the peaks above our dotted line show significant difference between the growth of the plasmids at these times. They were coded with rolling averages in 5 min increments up to 50mins.
• For 0mM:
  
  
  
• For 250mM:
  
  
  
• For 500mM:
  
  
  
At this ammonium concentration, all data values show significant differences between each copy number plasmid.

Sigmoidal Curve Fitting - & Fluorescence Rate Stats

• Using the sigmoidal function: L / (1 + np.exp(-k*(x-x0))) + b, we fit each of our growth curves to a sigmoidal curve to smoothen them out. As we have quite dramatic death phases, we gave the curve the max values of our data to stop fitting at.
• For 0mM:
  
  
  
  
  Here, there's a pattern of pBBR1, pSC101, pUC (low/med, low, high copy number) where midpoint increases (takes longer to reach half of its maximum fluorescence intensity), carrying capacity increases (greater maximum fluorescence intensity) and doubling time increases too.
• For 250mM:
  
  
  
  
  
  
  The pattern remains the same but the midpoints increase as fluorescence rate slows and carrying time decreases as ammonium decreases the max fluorescence.
• For 500mM:
  
  
  
  
  Carrying capacity decreases as copy number decreases, doubling time increases but pBBR1 has the lowest midpoint.
• For 750mM and 1000mM: No curves to fit.


Test 2- 08/09/22

Well Plate Decryption:



Growth Curves:

OVERVIEW: 3 JUMP DV’s Growth varying with [NH4]2SO4 concentration:


For all copy number plasmids, it appears that ammonium sulphate of any concentration is detrimental to growth.

JUMP DV’s & controls Growth per [NH4]2SO4 concentration:




• At 0mM, pUC, the highest copy number plasmid containing cells has the longest stationary phase and has the greatest amount of growth. This is followed by pBBR1 at med/low copy number, with a similar high growth but dies off quickly. pSC101 is the lowest copy number plasmid and has even less growth.
  These are the controls to compare the ammonium effects to.
  {From the paper, we were told to expect ammonium “impairs growth” as a “result of general osmotic or ionic effects.”}
  
• At 180mM [NH4]2SO4, all growths are effected but it looks like pBBR1 has a similar maximum growth as pUC, though pUC had a slower growth rate due to the metabolic burden of the high copy number.
• At 360mM [NH4]2SO4 pUC no longer has the greatest growthwhilst the lower copy number plasmids are effected similarly.
• 540mM [NH4]2SO4 effects the higher copy number plasmids worse but overall slows down the growth rate of all cells with them all only just finishing exponential phase at the end of the experiment.
• At 720mM [NH4]2SO4, it appears that this level as well as 750mM, is too high for the cells. All cells are unable to grow significantly under this high molarity of ammonium.


ANOVAs - Are we seeing significant differences between different copy number plasmids?

• Yes!!
• With our ANOVA plots, the peaks above our dotted line show significant difference between the growth of the plasmids at these times. They were coded with rolling averages in 5 min increments up to 50mins.
• For 0mM:
  
  
  We see 3 main peaks at stages of growth: exponential, stationary and death.
• For 180mM:
  
  
  We see large significances at exponential phase and as the cells go into death phase.
• For 360mM and 540mM:
  
  
  
  
  Growth seems to be significantly different all throughout the cells growth phases.
• For 720mM:
  
  
  
• There are no significance peaks here so all the cells are equally detrimentally affected by the high ammonium concentration.


Sigmoidal Curve Fitting & Growth Rate Stats

• Using the sigmoidal function: L / (1 + np.exp(-k*(x-x0))) + b, we fit each of our growth curves to a sigmoidal curve to smoothen them out. As we have quite dramatic death phases, we gave the curve the max values of our data to stop fitting at.
• For 0mM:
  
  
  
  
  
  As copy number increases midpoint increases (it takes longer for the cells to reach half of their maximum growth - copy number burden delays growth).
  BUT carrying capacity order goes pBBR1 < pSC101 < pUC and dt (doubling time) has the pattern pSC101 <pUC < pBBR1.
• For 180mM:
  
  
  
  
  
  The midpoint increases as copy number does here, and so does doubling time so the greater metabolic burden effects it growth in the presence of ammonium. pBBR1 has the greatest carrying capacity though so it is able to push through and still grow the most despite the ammonium despite how long it takes.
• For 360mM:
  
  
  
  
  Carrying capacity decreases with copy number at this high ammonium concentration - the cells may have reached their ammonium limit in terms of maximum growth.
• For 540mM:
  
  
  
  
  
  Carrying capacity decreases with copy number here too with the greatest doubling time for the lowest copy number plasmid.
• For 720mM:
  No curve to fit


Fluorescence Intensity Curves:
Here, the plasmids in the distribution kit contain sfGFP which is easy for us to measure. However, our contribution also has a greater significance as this sfGFP can stand in for any coding sequence that can be cloned into these destination vectors. For certain levels of expression, synthetic biologists can use our data to determine which copy number would suit their application best, and how osmotic/ionic effects are portrayed through the expression.

OVERVIEW: 3 JUMP DV’s Fluorescence Intensity varying with [NH4]2SO4 concentration:



For all copy number plasmids, ammonium sulphate of any concentration is detrimental to growth however:

pSC101 (low copy no.): Ammonium actually enhances fluorescence intensity/protein expression up to a point! Even though the fluorescence rate decreases, the maximum fluorescence increases as we increase ammonium up to 540mM (no fluorescence at 720mM - it’s limit).

pBBR1 (low-med copy no.): Ammonium actually enhances fluorescence intensity/ protein expression up to a point! Even though the fluorescence rate decreases, the maximum fluorescence increases as we increase ammonium up to 360mM (less fluorescence at 540mM, no fluorescence at 720mM - it’s limit).

pUC (high copy no.): For high copy number plasmids, it turns out that any ammonium concentration is detrimental to the cells expression! The greatest fluorescence was recorded with 0mM of ammonium sulphate, with decreasing expression as this was increased (no fluorescence at 720mM - it’s limit).

JUMP DV’s & control’s Fluorescence Intensity per [NH4]2SO4 concentration:




• At 0mM, pUC, the highest copy number plasmid containing cells has a significantly greater expression of sfGFP. This is to be expected as more plasmids = more sfGFP = greater fluorescence intensity.
• {From the paper, we were told to expect ammonium “impairs growth” as a “result of general osmotic or ionic effects.” so let’s investigate its effect on fluorescence}
At 180mM [NH4]2SO4, the higher the copy number plasmid, pUC fluorescence has been lowered slightly, but with minimal effect on the lower copy plasmids. The metabolic burden of having a higher copy number may be making the E. coli more susceptible to the ionic/osmotic effects.
• At 360mM [NH4]2SO4 the pUC fluorescence is lowered much more but pSC101 and pBBR1 seem to have increased their fluorescence.
• At 540mM, pUC fluorescence intensity increases at a slower rate, not even reaching its maximum during the experiment At 720mM [NH4]2SO4, no fluorescence is observed.


ANOVAs - Are we seeing significant differences in fluorescence between different copy number plasmids?

• Yes!!
• With our ANOVA plots, the peaks above our dotted line show significant difference between the growth of the plasmids at these times. They were coded with rolling averages in 5 min increments up to 50mins.
• For 0mM:
  
  
  Large significance at all stages of growth
• For 180mM:
  
  
  Large significance at all stages of growth
• For 360mM:
  
  
  Large significance specifically entering stationary phase
• For 540mM:
  
  
  Significance all throughout specifically entering stationary phase.


Sigmoidal Curve Fitting - & Fluorescence Rate Stats


Using the sigmoidal function: L / (1 + np.exp(-k*(x-x0))) + b, we fit each of our growth curves to a sigmoidal curve to smoothen them out. As we have quite dramatic death phases, we gave the curve the max values of our data to stop fitting at.

• For 0mM:




As copy number increases, the midpoints increase (takes longer for the cells to reach half of their maximum fluorescence), their carrying capacity and doubling time also increases.
• For 180mM:
  
  
  
  
  
  The pattern remains the same but for pBBR1, the doubling time is the lowest (doubled the fastest)
• For 360mM:
  
  
  
  
  
  The pattern remains the same again but for pBBR1, the doubling time is the lowest (doubled the fastest) AND it has the lowest carrying capacity - so the cells grew that fastest but weren’t able to fluoresce much.
• For 540mM:
  
  
  
  
  
  As copy number increases:
    Midpoint increases (it takes longer for the cells to reach half of their maximum growth - copy number burden delays growth).
    Carrying capacity increases (cells reach higher max. growth).
    dt (doubling time) increases (it takes longer for the cells to double - again, copy number burden)
• For 750mM:
  No curve to fit.

3. DIY Team T-Shirts

We think many teams can agree that iGEM is so much more than just a synthetic biology competition. iGEM encourages teams to develop a huge array of skills including outreach and communication, scheduling, planning and importantly in negotiating sponsorship. After going through the experience of seeking out sponsorships, it has become evident that luck is not always on our side despite our many attempts at reaching out to companies. Also with the fees of attending the Jamboree itself, we realise that not all teams will have the finances to allocate to the more ‘aesthetic’ parts of the project.
But iGEM is a community and having something as simple as team t shirts can make the experience just that more memorable. We would like to help alleviate some of the financial stresses of future teams by giving a simple guide to DIYing your own team t-shirts for cheaper!

You will only need to purchase ‘Iron on transfer Sheets’ which you can purchase on ebay for £1.99 (20 sheets) and a white/light coloured t shirt which you can get from ‘fruit of the loom’ for £2.51 and upwards. And as long as you have an iron and a printer you are all set!

Instructions:

1. Design your team logo on any software you are confident with. For our logo we used Inkscape.
2. Mirror your image - the image will be the right way round once transferred.

3. Export that image at a high quality onto Microsoft Word or a software that you can print from.
4. Print out the design on ordinary paper to check whether the colours are right or if you need to adjust the size.
5. You can also use this as a model to see whether you like the positioning/size of the logo by gently attaching it to the t shirt with a rolled up bit of cellotape (to make it double sided). This way you can get a general idea of what it would look like before it’s added permanently.
6. Once happy, print it on the transfer paper.
7. Cut around the logo - even if there blank space, the transparent film will still transfer there.
8. Place your t shirt onto an ironing board and iron it if wrinkled - you need a smooth working surface.
9. Place a piece of cardboard inside the t shirt to prevent it transfering through.
10. We recommend lighter t shirt colours as more designs will be visible!
11. Place the logo on the t-shirt, image side down, in the position that you want it - you may want to use a ruler and some chalk to get it precise.
12. Iron over the paper at 190°C for a few minutes, going over it multiple times.
13. Allow the paper to cool completely.
14. Remove backing.
15. There you have your very own custom t shirt!

We are using this on our t-shirts and sweatshirts after purchasing them with our team logo on to add sponsors logos, our names and our university logo to save money (and look good whilst doing so!).