Week One

Digestion and Isolation of pSB1C3 Backbone.

The purpose of digesting the pSB1C3/PArsRGFP construct was to separate the backbone from the former GFP insert. Tacoma RAINmakers sought to isolate the pSB1C3 backbone employed in their 2017 construct, as the GFP reporter complex was no longer desired. Apparent disadvantages of GFP indication in biosensors are ultraviolet readings. The RAINmakers prefered a chromoprotein that produces color in the visible spectrum. Enzymes XbaI and SpeI were used to cleave the terminator sites of the vector, freeing the pSB1C3 backbone. NEB resources confirmed that the Cutsmart Buffer 2.1 is the most compatible with these particular enzymes, providing an optimized environment for digestion. Combining the reagents listed in Table 1.0, the reaction was set at 37ºC in a water bath for 1 hour and 45 minutes. This reaction was completed in duplicate to increase statistical probability of desired backbone DNA.

Following completion of pSB1C3 digestion, Tacoma RAINmakers employed a standard procedure listed in the protocol page as “Agarose Gel Electrophoresis.” The purpose of this gel was to confirm if the backbone DNA had been successfully isolated from the undesired GFP insert. As cited in Figure 1.1, the expected pSB1C3 bands were located at 2000bp. A gel extraction process, also outlined in the protocol section, was completed in order to remove and contain the digested pSB1C3 DNA.

Week 2

Digestion and Ligation of spisPink, amilCP, and PcArsR Inserts

Using IDT stock solutions of chromoprotein and arsenic regulatory DNA, Tacoma RAINmakers completed a standard restriction digest (see protocol page for further information). The notable difference between insert and backbone digestion are the enzymes. Each insert included a SalI enzyme site, which is not compatible with the BioBrick suffix/prefix of the vector. Instead, the SalI site is used to ligate the chromoprotein to PcArsR, rendering the ends of this complete insert as compatible for sticky-end ligation to pSB1C3. After combining reagents listed in Table 2.0, reaction was held in a water bath at 37°C overnight.

Once the SalI site in spisPink, amilCP, and PcArsR was successfully stick-ended, Tacoma RAINmakers were prepared for ligation of each chromoprotein to the arsenic regulator. 60ng of each part were used in order to ensure that there would enough DNA material for the ligation. Having combined the substances from Table 2.1, the reaction was set to ligate overnight at 16°C. Afterwards, SalI was heat inactivated at 80°C, ensuring a complete denature, since the enzyme was no longer required.

Week 3

Digestion and Ligation of Insert (amilCP/spisPink + PcArsR) to pSB1C3 Backbone

Following the ligation of the chromoprotein and PcArsR, the complete insert was digested with enzymes compatible with our pSB1C3 backbone. This process allows sticky-ended ligation in the next step, which increases the chance of a proper insert-backbone ligation. 84 ng of insert DNA was pipetted into the reaction alongside the other reagents mentioned in Table 3.0. The digestion was set at 37°C in a water bath for 1 hour and 30 minutes.

Tacoma RAINmakers combined the reagents listed in Table 3.1 to ligate the completed insert to the vector. The reaction included a negative control that contained only vector DNA. A notable process involved in ligation reactions is calculating DNA volumes. Typically, a 1:3 ratio of vector to insert ensures that there is a balance of both parts. The RAINmakers employed the NEBioCalculator to determine how many moles were in 1ng of vector (2070bp) and 1ng of insert (1488bp). This calculation translated to 0.8µL of vector and 20µL of insert. The ligation occurred at 16ºC overnight and was heat inactivated at 80ºC for 20 minutes the following morning.

Initiation of Individual Chromoprotein and Regulator Plasmid Design

A standard procedure in the Tacoma RAINmakers project is PCR amplification. This process is listed under the protocol page as “Insert PCR Amplification.” In preparation for ligation of inserts (amilCP, spisPink, and PcArsR) into the vector, all insert DNA must be amplified from its original limited stock. Once the PCR reaction has exited the thermocycler, gel electrophoresis must be employed to assess the efficacy of the amplification. As pictured in Figure 3.2, both the spisPink and amilCP bands successfully appeared at about 1000bp, and the PcArsR expressed at about 550bp. Unfortunately, the PcArsR negative control produced DNA bands, which suggested contamination during the PCR amplification process.

Following a successful PCR amplification confirmed by the gel, Tacoma RAINmakers performed a standard gel extraction. With much more insert DNA, RAINmakers were prepared to design three additional plasmids containing single inserts for isolated testing.

Week 4

Transformation of Ars3.0 Construct and PCR Screening

TThe positive control (pSB1C3 + Insert) and negative control (pSB1C3 only) were transformed using DH5 Alpha Competent E. coli cells. Although the iGEM protocol for transformation states that 1µL of DNA is sufficient, 2µL of DNA were used for both the positive and negative control plates to ensure enough DNA existed for multiple colonies to grow. Transformation reactions were incubated overnight at 37ºC within a 14-18 hour time period. An extended description of the standard transformation process is listed under the protocol page.
The RAINmakers ran a PCR screening for both the positive (vector and insert) and negative (vector only) controls. This PCR allows us to amplify our cloned DNA, which is necessary to do because it will help us determine whether or not the ligation of the vector and insert was successful. The product after our PCR will be used to run a gel, which will help us see which colonies have the vector and insert and which only have the vector. After determining the volumes of the reagents, we decided to add an extra 20% uncertainty to each reagent in the master mix since small volumes of liquid can get stuck in the pipette. We did all these calculations based on the fact that we planned on doing 8 PCR reactions (8 PCR tubes). Before beginning the process of adding all the reagents to our PCR tubes, we needed to determine how long the extension part of PCR should be based on the base pair count of our product. Running a PCR simulation of Snapgene allowed us to find the exact base pair count of our positive control, which is 1597 base pairs. From this, we found that the extension period should be 1 minute and 36 seconds.
After adding all of our reagents together into 8 PCR tubes, we needed to select single colonies from transformation plates from 6/19/18. We determined that 6 of our PCR tubes would have the positive control, 1 would be the negative control and the last tube would have no DNA. Once we selected a single colony and put the DNA into a PCR tube, we streaked it onto a new plate (cut up in 8 different sections for each 8 different colonies) and labeled them 1,2,3,4,5,6 (positive control), 7 (nothing) and 8 (negative control). We then incubated this new plate so that more colonies would grow. Finally, we put our PCR tubes in a thermocycler and ran our PCR.

Blunt-End Digestion and Ligation of pSB1C3 and Inserts

Designing plasmids with individual inserts becomes slightly complicated, as each insert contains a SalI site that is not compatible with the BioBrick prefix/suffix of the backbone. Hence, Tacoma RAINmakers preferred blunt-end ligation, rendering the incompatible ends irrelevant. Table 4.0 outlines the reagents that were added to the pSB1C3 backbone DNA to create blunt ends.
Additionally, each insert was digested with its respective enzyme and filled in with T4 polymerase in preparation for blunt-end ligation.

Week 5

not updated yet

Week 6

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