Engineering
Summary
We were able to successfully assemble all of our planned constructs with the exception of pSB1C3::CcdA::CcdB by analyzing the mode of failure and redesigning our cloning protocol to resolve identified issues.
For expression verification of our proteins, we created a simple and novel thermolysis protein extraction protocol as a means of overcoming equipment limitations, then successfully demonstrated the viability of this protocol. We resolved clumping issues during sample preparation and used SDS-PAGE to demonstrate that samples treated using this protocol produce interpretable and viable results.
For a comprehensive experimental overview of our project, please visit the Experiments page..
Cloning
Plasmid inserts were acquired by ordering the synthesis of our construct designs via IDT gblocks synthesis.
Vectors were prepared by digesting pSB1C3::BBa_J04450 then performing gel extraction on the 2k bp band. Inserts were digested and then DNA-extracted without running gel electrophoresis, as the sequence fidelity of synthesized gene fragments is high. Quantification was performed on the extracted DNA, and calculations were made to approximate the 3:1 insert-to-vector ratio. The ligation product was transformed into DH5α; after heat shock and six minutes of incubation on ice, the mixture was then plated on LB plate supplemented with chloramphenicol for selection.
Cloning procedures for the chi18h8 and mleR construct were broken up into two stages due to errors during gblocks synthesis. The process is explained in the figure below.
Figure 1b. Two-step cloning process for pSB1c3::mleR
Apart from excess colony number on some plates, plating results appeared visually normal for the most part during cloning for pSB1C3::chi18h8(1/2), pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2).
Colony PCR
Colony PCR was performed using backbone-specific primers; they attach to the biobrick prefix and suffix sites to flank inserts. The results of PCR should thus be DNA bands with the same length as the insert.
Ten colonies were chosen over multiple colony PCR tests, and pSB1C3::BBa_J04450 is used as the negative control. Unfortunately, the vast majority returned as negative. Multiple double PCR bands were observed, and there was a consistent and anomalous 1.5k bp band visible on many PCR results. Four of the chi18h18(1/2) (labeled “chi”) colonies displayed the correct ~800 bp band corresponding to chi18h8(1/2); the rest were entirely incorrect. We initially hypothesized this result to be caused by antibiotic degradation decreasing the effectiveness of the selection plate.
Additional cloning cycles were performed to create pSB1C3::chi18h8 (the two step cloning process illustrated in Figure 1a.), pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2), this time with freshly cast LB agar plates. Plating results for the chi18h8 yielded numerous colonies, but poor colony PCR positive rate - 1 out of 11 displayed the 1.8 kb insert band corresponding to the correct and complete chi18h8 construct.
The singular verified DH5α pSB1C3::Chi18h8 colony was grown in liquid culture; miniprep was then performed to extract pSB1C3::Chi18h8.
Results for the other three constructs were exceptionally poor; only two colonies grew on the pSB1C3::CcdA plate and one for pSB1C3::CcdB and pSB1C3::mleR. All PCR bands were identical to the negative control (BBa_J04450).
Troubleshooting
Cloning cycles for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2) failed. The erratic quantity of bacterial growth was inconsistent with digestion or ligation failure. Analysis of the failure mode demonstrated that failed bands appear to be of erratic, inexplicable sizes (1.5 kb, < 1 kb) that did not correspond to negative control pSB1C3(2k bp) or BBa_J04450(1k bp) bands.
We thus hypothesized the underlying issue to be poor transformation efficiency resulting in low numbers of successfully transformed bacteria. To resolve this, we chose to include a recovery step by incubating the transformation mixture in LB to increase the number of viable bacteria.
Re-attempt
Cloning cycles for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR were redone with the additional step of recovery. The colony quantity on the plates appears visually normal.
Re-attempt Colony PCR
Five colonies were chosen from each plate for colony PCR verification; pSB1C3::BBa_J04450 was again used as the negative control. No dual bands were observed, and the success rate was significantly higher. Four of the pSB1C3::CcdA bands matched with the ~300 bp insert size, at least 3 of the pSB1C3::CcdB bands matched with the ~400 bp insert size, and all of the pSB1C3::mleR(1/2) bands matched with the ~600 bp insert size.
The massive improvement in success rate after choosing to perform recovery demonstrates the effectiveness of the method and the validity of our hypothesis.
All verified construct colonies were then grown in liquid culture for plasmid amplification; miniprep was then performed to extract the constructs.
The redesigned protocol was applied to successfully assemble the following constructs: pSB1C3::CcdA, pSB1C3::CcdB, pSB1C3::mleR, pSB1C3::Chi18h8, pSB1C3::CcdA::mleR. These constructs were then transformed into BL21(DE3) for expression.
Protein Expression/Thermolysis
Chi18h8 expression was performed in 50 ml of expression medium with BL21(DE3) E. coli. During literature research, it seemed apparent that expression of the protein required very particular protocols; it was thus decided that we’d follow Berini et al.’s protocol at all points if possible. Chi18h8 expression was performed at two temperatures, 37 oC and 30 oC; the former was used as a reference for standard in vitro settings, and the latter was used to simulate soil temperature. 5 ml of expression medium was used for cell harvest at time intervals of 2h and 4h. For CcdB and mleR, cell harvest was performed at time intervals of 0h, 0.5h, 1h, 2h, 3h, and 4h. For CcdA, cell harvest was performed at 3h. Expression protocol mostly followed Berini et al.’s methods.
Thermolysis
However, we hit a roadblock at protein extraction due to a lack of equipment for performing sonication and chemical lysis. To resolve this, we formulated a thermolysis method based on tips from Lab 106 personnel and theory. Phosphate Buffered Saline (PBS) and 4X Sample buffer were added to pelletized cells in a volume ratio of 3:1, the total fluid volume was initially adjusted to 100 ul, and the cells were resuspended. This process was performed on CcdB and mleR samples.
It was noticed immediately that for larger pellets, the resuspended mixture became highly viscous, and clumping was observed in some cases. After heating the mixture at 95oC for 10 minutes, centrifuging at 4oC, 16000g, the supernatant was extracted and transferred to a new eppendorf tube; in multiple cases, a clump was left over. This also had the effect of significantly impacting the supernatant yield - only a few bubbles could be extracted from the thermolysed cell mixture.
Troubleshooting
After consulting our instructor on this odd occurrence, she suggested it could be due to high concentrations of genomic DNA resulting in clumping. To resolve this issue, we decided moving forward to use double the total volume, i.e. 200 ul, to resuspend pellets. We hypothesized that this should dilute the genomic DNA and reduce clumping.
Re-attempt
Protein samples that underwent clumping were salvaged with 100 ul extra PBS, then reheated to 95 oC for 10 minutes to break up the clump.
The heated eppendorf tubes were inspected mid-heating, and the clumps were observed to have somewhat dissolved. After the full 10 minutes, the clumps mostly disappeared. The mixture was re-centrifuged, and the supernatant was again transferred to a new eppendorf tube.
SDS-PAGE
20 ul of the extracted proteins were taken and analyzed via SDS-PAGE.
For both mleR and CcdB, correct protein bands are visible to a reasonable degree.
Chi18h8 and CcdA-mleR samples were thermolysed with the new 200 ul resuspension volume protocol, and essentially no clumping occurred.
For Chi18h8, protein bands were obscured by excessive protein quantity and excessive dyeing, but they are nevertheless distinguishable. For CcdA-mleR, protein bands were no longer distinguishable below 17 kDa; however, we believe this to be caused by the buffer system we were using. Because Tris-Glycine is poor at resolving protein bands smaller than 15 kDa, it is natural that CcdA(9.5kDa) would be hard to distinguish. Apart from that, correct, distinct mleR bands at ~35kDa were visible throughout all trial groups.
The extracted proteins were used for expression verification only due to time constraints. Since SDS-PAGE analysis demonstrates that proteins of interest were successfully extracted via thermolysis, we are able to conclude that our protocol for thermolysis is viable as a method of protein extraction for expression verification purposes.