Apart from the Crafting Crocin main project, the iGEM Uppsala 2017 team decided to incorporate a somewhat unrelated side project – Chipgineering 2.0, a continuation of the iGEM Uppsala 2016 Chipgineering project. We did this because some team members felt a strong desire to carry on the legacy of the 2016 Team in some form and as it was felt we had a large enough team to accommodate this desire, the side project was given a go-ahead. With the blessing of our team, we, the side-project group forged ahead with the ambition to improve upon last year's design. The design we decided to improve was the heat shock transformation chip, since the transformation chip had shown results when tested by last years team. But, there were still technical problems, such as how heat was supplied to the chip. Therefore, we saw this as the main way of improving the heat shock chip.
Design
Having decided to focus on heat shock, the first task for our group was to redesign the actual chip. AutoCAD and Rhino 3D were used to redesign the negative template for molding the polydimethylsiloxane (PDMS). This involved changes to some of the dimensions to optimize the heat transfer for the transformation reaction. The redesign also served to adjust the inlet/outlet, the transformation channel, and the thickness of the PDMS chip itself. An early iteration can be found in figure 1, and figure 2 shows the final design that was used. When the design was finished, the mold could then be printed by using resin 3D print.
Figure 1. Example of an early chip model in Rhino 3D Figure 2. Final design of negative mould used for experiments.
Considering the inefficient heating process of the previous heat shock transformation chip, this year we created an Arduino module which can generate the specified heat. The module contains an Arduino microcontroller, a temperature sensor, a mini LCD monitor, a peltier, a mosfet, LED and an adaptor which can produce 9 V with 2.25 A. The module was assembled according to figure 3 and programmed with a Arduino program (code found here). To test whether the code worked a test was conducted by aligning the temperature sensor, PDMS chip and peltier. A stopwatch was used to measure the heat transfer within the chip in real time. Within less than 70 seconds, the heat stabilized the PDMS chip temperature which was measured by the temperature sensor. Should our final design prove successful, we were planning to change the energy sources (by using battery) to make the whole transformation chip more portable.
Figure 3a The heat source optimization and the module for peltier control in arduino: Heat transfer test. Figure 3b The heat source optimization and the module for peltier control in arduino: Arduino module which used to control the peltier (heat source) (modified from (1))
Result
Having created the new designs the next step was to print and test them. The new designs differed from our predecessors in that we bonded the PDMS to glass by using a corona discharger instead of sandwiching the PDMS between plexiglass sheets. We also switched from a straight to a serpentine channel (figure 4 and 5) and eliminated the on chip-heating channels by using the peltier element. Figure 4: AutoCAD model of the chip. The red box is the peltier, the grey object is the glass slide and the yellow object is the PDMS chip. While we had planned to carry out a battery of experiments to test the chip, in the end, we only had time to carry out one of them: testing transformation efficiency. As outlined below, it did not succeed as well as we had hoped.
The Experiment
The experiment in brief was to carry out heat-shock transformation on the chip depicted in figure 5 using the final design (figure 2) combined with a peltier-element as illustrated in figure 4.
Figure 5: Assembled microfluidic chip with needles and some tubing.
The experiment was set up into four different parts:
Perform regular heat shock transformation
Add DNA but no heat shock is performed
Heat shock without adding any DNA
Heat shock through the chip with DNA added, collected in four tubes.
And executed according to the protocol found here. Results of these experiments are shown in table 1.
The chip did not perform at all, and there is also an anomalous result with the cells that were not heat shocked forming a greater number of colonies than the heat shock transformed ones, where you would expect less colonies or none at all. As we did not have time to perform any replicates or additional experiments it is hard to draw any conclusions from these results. The anomalous colony forming may well be a result of improper mixing of antibiotics during plate making or errors in handling the tubes. We also did not have time to optimize the transformation protocol and setup of the chip, which may have led to the failure to transform.
References
Arduino/Microcontroller MOSFET [Internet]. Instructables.com. [cited 2017 Oct 30]. Available from: http://www.instructables.com/id/ArduinoMicrocontroller-MOSFET/
