Results from our 2023 BloomBiota wet lab experiments
______________________________________________________________________
Extraction and confirmation of Plasmid pBeloBac11loxP2272 from stab innoculation
pBeloBac11loxP2272 was grown in E. coli multiple times for extraction using a miniprep kit with mixed results (Table 1). The LB media used to culture the E. coli was supplemented with 25 μg/mL chloramphenicol for the selection of positive transformants. Transformations were confirmed using BsaI HF-v2 restriction enzyme and gel electrophoresis on a 0.8% agarose gel. The low copy plasmid had better extraction results when more culture was centrifuged prior to plasmid extraction, with 180.7 ng/μL being the best result from any of the extraction attempts. Confirmation of this extraction was not successful following digestion. However, after PCR with appropriate primers, the band appeared at the correct place on the gel, confirming that the plasmid extraction was successful.
Table 1: Summary results of the attempts to extract and confirm the pBeloBac11loxp2272 plasmid (7,507bp) from E. coli.
| Miniprep success | Concentration (ng/μL) | A260/A280 | Notes | Date(s) |
|---|---|---|---|---|
| Half success (2 tubes) | 24.8 30.7 |
Not recorded | Miniprep #1 was confirmed on a gel, but #2 was not. | July 3, 2023 |
| Success (1 tube) |
180.7 | Not recorded | There were no bands on the gel, but PCR was done on the sample, and PCR successfully amplified the plasmid. |
July 6, 2023 |
| Success (5 tubes) |
35.8 77.2 74.9 45.8 58.6 |
2.01 2.05 2.07 2.06 2.04 |
The miniprep kit was from 2019, as the previous kit ran out of reagents. DNA purity ratio indicates likely RNA contamination in minipreps |
July 13, 2023 |
______________________________________________________________________
PCR of fragments for Gibson Assembly
PCR of the vector and vitamin B12 gene fragment was attempted multiple times, with results summarized in Table 2. Vector pBeloBac11loxP2272 was ordered from AddGene and extracted from E. coli prior to PCR. Gene fragments CbiA-H and CbiJ-CobT were PCR’d from Salmonella Typhimurium LT2 gDNA. The success or failure was determined via performing electrophoresis on a 0.8% agarose gel. The first attempt failed for all three amplicons, although subsequent attempts were successful and generated high amplicon concentrations.
Table 2: PCR results of the three fragments needed for Gibson assembly of the Vitamin B12 gene cluster.
| Genes CbiA-H (ng/μL) | Genes CbiJ-CobT (ng/μL) | Vector (ng/μL) | Notes | Date(s) |
|---|---|---|---|---|
| Fail | Fail | Fail | PCR was not successful | July 4, 2023 |
| 451.5 | 467.0 | N/A | Vector PCR was run on gel a second time, and failure was confirmed again. CbiA-H (2 lanes, separate reactions) both bands at ~8.0kbp, slightly higher than expected. CbiJ-CobT (2 lanes, different reactions) had one band at ~4kbp and a second at ~9.5kbp. Gel was on a slight slope when solidifying, thicker at the positive electrode and slightly thicker from lanes 1 to 8. | July 5, 2023 |
| N/A | N/A | 375.6 | Vector PCR was successful with the band at the correct place (~7.55kbp) on the gel after PCR of miniprep that was not gel confirmed. | July 7, 2023 |
| 454.7 Clean = 72.7 |
557.6 Clean = 64.6 |
393.6 Clean = 116.7 |
PCR of fragments redone before another attempt at Gibson Assembly. Bands at expected regions on the gel. CbiA-H band is an odd smile-shaped band. PCR amplicons were cleaned with a PCR cleanup kit in case leftover reagents affected the Gibson assembly. A gel run to confirm the presence of amplicons after PCR cleanup was positive for all three amplicons. | July 24, 2023 July 25, 2023 |
| Fail | N/A | An attempt to amplify the entire vitamin B12 gene cluster was not successful. A large number of bands from 100bp to 500bp with another band at ~3.8kbp. | Aug 14, 2023 | |
| Fail | N/A | A second attempt to amplify the entire vitamin B12 gene cluster using different reagent conentrations was not successful. Bands appeared from 100bp to 12+ kbp, but no bands at ~16kbp. | Aug 16, 2023 | |
Figure 1: Gel Electrophoresis Confirmation of PCR Products After Cleanup
Note: A 0.8% agarose gel was run for 60 minutes at 100V. PCR products for Gibson Assembly after amplicons were cleaned up with a PCR cleanup kit to remove potential contaminants. The bands are around their expected locations: 7.6kbp for vector, 7.9kbp for CbiA-H, and 8.7kbp for CbiJ-CobT.
______________________________________________________________________
Gibson Assembly of Vitamin B12 Gene Cluster
Gibson Assembly of three fragments was attempted multiple times, with results summarized in Table 3. Three DNA fragments with overlaps produced via PCR were used in all attempts, and transformation was performed directly after incubation of assembly. White/blue colony screening with X-gal media was initially used to determine which colonies to screen (white ones) for successful insertion by performing electrophoresis on a 0.8% agarose gel after digestion with BsaI HF-v2. All transformations were performed with NEB alpha-competent cells, dh5α derived.
The default 15-minute incubation time was negative for any colonies. Attempts 2 through 7 all produced white colonies after transformation on plates with 25 μg/mL chloramphenicol. Following successful colony formation, Gibson Assemblies were confirmed with digestion, gel electrophoresis and PCR. Using primers designed to create amplicon fragments for assembly, new PCR primers were ordered to confirm Gibson Assembly attempts 2, 3, 4, and 5. All attempts at confirmation failed.
Table 3: Summary of results from experiments to use Gibson Assembly to construct a plasmid containing the vitamin B12 gene cluster controlled by a constitutive promoter.
<| Attempt Number | Incubation time | Notes | Result | Date(s) |
|---|---|---|---|---|
| 1 | 15 minutes | The default time (15 min) for Gibson Assembly protocol was followed. No colonies formed. | Negative | July 10, 2023 July 11, 2023 |
| 2 | 60 minutes | A single white colony formed on one plate after ~24 hours of growth. The fire alarm went off three times during the experiment; X-gal may not have been appropriately spread due to this, as it formed a whitish patch on the agar after the team was forced to evacuate before the X-gal could be spread around the plate. No bands on the gel after BsaI HF-v2 digestion. Attempting colony PCR and PCR the miniprep of the white colony for insert confirmation using the same primers used to create the inserts also failed. Three sets of primers, each flanking an area where two fragments join, were used to confirm Gibson Assembly; no bands were seen for any set of primers. | Negative | July 11, 2023 July 12, 2023 July 22, 2023 |
| 3 | 75 minutes | A single white colony formed. Transformation protocol confirmed as a positive control using unaltered plasmid grew ~80 blue colonies. No bands on the gel after BsaI-HF v2 or EcoRI digestion. Attempts to colony PCR and PCR miniprep of the white colony to confirm the insert using the same primers used to create inserts also failed. | Negative | July 13, 2023 July 14, 2023 July 18, 2023 July 22, 2023 |
| 4 | 3.5 hours | 3 white colonies on one transformation plate. No bands on the gel after EcoRI digestion. Attempts to colony PCR and PCR miniprep of the white colony to confirm the insert using the same primers used to create inserts also failed. CbiA-H fragment confirmed to be in assembly mix via PCR of mix, CbiJ-CobT fragment not amplified. All three colonies were tested with negative results for all. Three sets of primers, each flanking an area where two fragments join, were used to confirm Gibson Assembly; no bands were seen for any set of primers. | Negative | July 24, 2023 July 25, 2023 July 30, 2023 Aug 1, 2023 |
| 5 | 60 minutes | PCR amplicons after PCR cleanup were used. 2-5 white colonies each, on three plates. No bands on the gel after EcoRI digestion. Attempts to colony PCR and PCR miniprep of the white colony to confirm the insert using the same primers used to create inserts also failed.biA-H fragment confirmed to be in assembly mix via PCR of mix, CbiJ-CobT fragment not amplified. Three separate colonies were tested, all with negative results. Three sets of primers, each flanking an area where two fragments join, were used to confirm Gibson Assembly. One band of the expected size for primers flanking CbiH-J genes was seen, but no other bands were found after two attempts for other primers. |
Negative | July 30, 2023 Aug 1, 2023 Aug 24, 2023 Aug 29, 2023 |
| 6 | 30 minutes and 30 minutes | Sequential Gibson assembly starting with vector amplicon and CbiJ-CobT amplicon before CbiA-H amplicon was added after 30 minutes incubation was negative with no colonies on the plate after transformation and 48 hours of agar plate incubation in 37℃ incubator. | Negative | Aug 20, 2023 |
| 7 | 40 minutes and 40 minutes | Sequential Gibson assembly starting with CbiA-H and CbiJ-CobT amplicon before vector amplicon was added after 40 minutes incubation was negative as all colonies (3-7 per plate) were blue. | Negative | Aug 27, 2023 |
______________________________________________________________________
Golden Gate Assembly of hGIF into pGGAselect
A Golden Gate Assembly BsaI-HF v2 kit from New England Bioscience was used to insert the hGIF into the pGGAselect plasmid before transformation in competent E. coli. Despite three separate attempts at insertion, with 7 different incubation times, followed by transformation, the experiment was not successful (Table 4). NEB α-competent cells were used for all transformations, forming colonies on attempt numbers 2 and 3. E. coli Nissle 1917 competent cells were used in attempts 2 and 3, with some colonies growing after attempt 2 but no colonies from attempt 3.
Table 4: Summary of experimental attempts to insert hGIF into pGGAselect using Golden Gate assembly with BsaI HF-v2 restriction enzyme were performed with no positive results.
| Attempt Number | Incubation time | Notes | Result | Date(s) |
|---|---|---|---|---|
| 1 | 5 minutes | After transformation onto chloramphenicol-containing media, multiple colonies grew on multiple plates after 24 hours in the 37℃ incubator. Seven colonies that appeared first, based on size, were selected for miniprep and digestion with EcoRI after overnight growth, with all seven isolates returning negative results. Miniprep DNA purity was bad for all minipreps with A260/A280 rations ranging from 1.27 to 1.44 with DNA concentrations ranging from 3.8 ng/μL to 65.6 ng/μL. Based on the band size (only for colonies 1, 2 and 3), the unaltered plasmid was transformed into the competent E. coli cells, no bands showed for colonies 4, 5, 6, or 7. | Negative | Aug 8, 2023 |
| 2 | 15 minutes | After transformation onto chloramphenicol-containing media, multiple colonies grew on multiple plates and 24 hours in the 37℃ incubator. Seven colonies that appeared first, based on size, were selected for miniprep and digestion with EcoRI. Miniprep DNA quality was poor, with colonies 1-3 and 5-7 having A260/A280 ratios ranging between 1.09 to 1.43 and DNA concentrations ranging from 19.0 ng/μL to 32.7 ng/μL. Colony 4 had aberrant results with an A260/A280 ratio of 2.14 and a DNA concentration of 275.7 ng/μL. Two of the expected bands were visible on the gel, the band at 100bp was not, with the lower band at ~1.4bp being very smudged. The colony and miniprep (#6) with the sharpest lower band was sent to plasmidsaurus for sequencing, but the results were negative as the returned sequence was the unaltered plasmid (no insert). Miniprep was attempted a second time on colony #6 overnight (new culture) but was unsuccessful with a bad A260/A280 ratio of 0.96 and DNA concentration of 21.7 ng/μL. Using different reagents, a third attempt to miniprep two separate tubes of colony #6 culture obtained good results with A260/A280 ratios of 1.94, 1.99 and DNA concentrations of 112.0 ng/μL, and 91.7 ng/μL. Results were negative on the gel after digestion and gel electrophoresis of miniprep number three. An attempt was made to transform the Golden Gate reaction into E. coli Nissle 1917, but was not successful and miniprep of transformants after overnight growth in broth did not have bands at expected sizes, though it did have larger bands at ~5kbp and ~6.5kbp | Negative | Aug 13, 2023 Aug 14, 2023 Aug 15, 2023 Aug 18, 2023 Aug 30, 2023 |
| 3 | 20 minutes 30 minutes 40 minutes 50 minutes 60 minutes |
High throughput Golden Gate assembly had 100+ colonies on plates from each incubation time (NEB alpha-competent cells but no E. coli Nissle 1917 cells). Some colonies were restreaked on new plates to ensure the antibiotic was still functional on the original plates and this was confirmed as those restreaked colonies grew without issue on new plates with chloramphenicol added. After overnight growth of two selected colonies from each incubation time, miniprep was done with concentrations ranging from 35.7 ng/μL to 69.4 ng/μL (one 60-minute colony was a slight outlier at 138.9 ng/μL) and A260/A280 ratios ranging from 1.89 to 2.01 (same 60-minute colony has the highest ratio at 2.07). Gel confirmation was inconclusive, with a band at ~3.4kbp and a band at ~2kbp/ | Inconclusive | Sept 15, 2023 Sept 16, 2023 Sept 20, 2023 |
Figure 2: Gel Electrophoresis Confirmation Gel From Golden Gate Assembly Attempt Two
Note: A 1% agarose gel was run for 35 minutes at 100V. Six colonies from Golden Gate assembly attempt two were grown overnight in brother before the plasmid was extracted with a miniprep kit. Colony four did not get plasmid extracted properly and was not digested. Bands would be expected at 2.1kbp, 1.4kbp and 33bp based on Benchling virtual digest with EcoRI. No band on any lanes at 33bp, but a sharp band at 2.1kbp and smeared bands from ~1.3-1.9kbp in lanes 2, 3, 5, 6, and 7.