We successfully amplified truncated fragments of ETR1 and EnvZ from A. thaliana cDNA (provided by Patrick Gulick, PhD) and E. coli genomic DNA (provided by Michelle Harvey, Tech). These truncated fragments will be used to generate site specific fusion proteins.

Overlapping PCR was attempted using a troubleshooting technique. Fragments used came from E.coli (EnvZ) and A. thaliana (ETR1). One nanogram of ETR1 and EnvZ fragments, corresponding to their proper fusion domains (DOM – domain, CON- conserved, and LIT-literature), were loaded using the standard contents necessary for PCR minus the primers. Four differing DNA polymerases were tested; PhireHot Start Polymerase, Phusion polymerase, Crimson Taq polymerase, and Q5 polymerase. Primers were added after 10 cycles out of a total of 30 cycles. PCR samples were then verified using a 1% agarose gel containing EtBr, and tested against ETR1 and EnvZ fragments (positive controls). Electrophoresis was conducted for 50 minutes at 100volts. Visualization revealed that out of the four polymerases used, only one was able to generated the correctly sized fusion proteins; Phire Hot Start DNA polymerase.

Any further purification of samples was suggested to be done using an ethanol purification protocol. Previously amplified Nested Deletion EnvZ, and Fusions proteins DOM and CON were Ethanol purified. Ethanol purification was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. Ethanol Purification products were then verified and quantified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Concentrations, determined through gel quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion. Additionally, amplification PCR for ETR1 and LIT Fusion were attempted again but were unsuccessful.

Temperature Gradient Amplification PCR was attempted for the ETR1 and Literature Fusion, since they were proving difficult to amplify. PCR was conduct using standard components, including specific primers, and 1 ng of Lit Fusion DNA, and 3ng of ETR1 cDNA. A positive control of EnvZ was also run in order to rule out potential discrepancies. The temperature gradient was set from 58 degrees Celsius to 62 degrees Celsius, and number of cycles was 35. Additionally, normal amplification PCR was conducted for 200-600 pg of Plac, and 1 ng of PompC-GFP DNA using standard protocol. Products were then tested using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization of the temperature gradient revealed that ETR1 products were amplified at the correct size for temperatures 60 to 63, and Lit fusion products were amplified correctly at 62 and 64. Also, Plac and PompC-GFP were also amplified properly.

Troubleshooting PCR amplification of; interface genes RhlR, LuxR, and LuxI was conducted. RhlI was used as a positive control since its amplification had been successful in numerous other PCR reactions. Standard PCR components, including specific and correct primers, were added to 200-600 pg of target DNA. PCR reactions tested common troubleshooting techniques such as; reduced primers, DMSO, added Magnesium content, and Taq polymerase. Ethanol Purification of DOM, CON and LIT fusions as well as Plac was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. PCR products were then verified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Out of the four parameters tested (plus one normal reaction); only added magnesium gave a visible result for LuxI and Lux R. However, the bands obtained were weak, therefore this reaction must be attempted again.

Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tested for PompC using restriction digest and PCR amplification. Standard PCR components, including specific and correct primers, were added to ~1ng of target DNA. Restriction Digest was conducted using standard Biobricks cut sites. PCR products and Restriction digest products, were visualized using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization revealed that PCR samples yielded a positive result for PompC-GFP in mini-preped samples, while restriction digest products also cut out a fragment the same size as PompC-GFP.