Experiments

Three E. coli backbone BioBricks (pBS1C, pBS2E, and pBS4S) and promoters (Pveg, PliaG, and PlepA) were miniprepped and used to create glycerol stocks.^2,3,4

B. subtilis was rehydrated and cultures were continually restreaked and maintained throughout the course of the project.

The two E. coli backbones and three E. coli promoters underwent restriction enzyme digestion and ligation to create six ligation combinations: pBS1C + Pveg, pBS1C + PliaG, pBS1C + PlepA, pBS4S + Pveg, pBS4S + PliaG, pBS4S + PlepA ^2,5,6

The pBS1C + Pveg ligation was transformed into competent E. coli cells.⁸ DNA gel electrophoresis was performed to verify successful ligation products, which were then replated.⁷

The remaining five ligation combinations (pBS1C + PliaG, pBS1C + PlepA, pBS4S + Pveg, pBS4S + PliaG, pBS4S + PlepA) were transformed into competent E. coli cells.^2,8 DNA gel electrophoresis was performed to verify successful ligation products, which were then miniprepped, replated and used to create glycerol stocks.^3,4,7

The miniprep products of the six verified ligation products (i.e., pBS1C + Pveg, pBS1C + PliaG, pBS1C + PlepA, pBS4S + Pveg, pBS4S + PliaG, pBS4S + PlepA) were quantified.

The pBS1C + Pveg ligation and MFS transporter plasmids underwent restriction enzyme digestion and ligation, eventually being transformed into competent E. coli cells. ^5,6,8 Colony PCR was performed for ten colonies to verify successful ligation products.⁹ Three successful ligations were identified and miniprepped.³

The miniprep products of the three successful pBS1C + Pveg + MFS ligations were quantified.

The MFS transporter plasmid was transformed, liquid cultured, miniprepped, and quantified.³

The remaining five verified ligation products underwent restriction enzyme digestion and ligation.^5,6The ligations were transformed into competent E. coli cells.⁸

Colony PCR was performed for five colonies of each of the ligations to verify successful ligation products.⁹Successful ligations were identified, miniprepped, and quantified.³

B. subtilis was prepared for electrocompetization - a first attempt was made to transform the six backbone-promoter-MFS ligations but the electroporator arced and killed the B. subtilis.¹⁷

More B. subtilis was prepared for electrocompetization with some adjustments to the protocol.¹

A second attempt was made to transform the linearized backbone + promoter + MFS ligations into the electrocompetent B. subtilis, but the electroporator still arced.¹⁷

Chemically competent B. subtilis was prepared and the same transformations were attempted, but the bacteria did not grow on the plates. ¹⁸

More successful ligations of backbone + promoter + MFS were miniprepped.³

Chemically competent B. subtilis was prepared a second time with modified conditions, and the same transformations were attempted.¹⁸ A lawn of bacteria grew and they were inoculated into liquid cultures containing chloramphenicol for pBS1C backbones and spectinomycin for pBS4S backbones. Nothing grew, confirming our hypothesis that they were not transformed.
A third attempt was made to chemically competize and transform B. subtilis using the Bacillus Genetic Stock Center protocol. However, some transformations either did not grow or formed a film. A third attempt was made to electrocompetize B. subtilis, and the DNA linearizations were PCR purified before transformation.^11,17 Although some colonies grew, the negative control was contaminated so we disregarded these results.

Chemically competent B. subtilis was made again and the growth was more carefully monitored.¹⁸ All the promoter + backbone + MFS ligations were transformed and some grew. Colony PCR was performed once but the liquid cultures grew very slowly.⁹ Colony PCR was performed again and none of the electrocompetent or chemical competent B. subtilis worked.⁹

We ordered supercompetent B. subtilis to retry the transformation.

Mannitol supercompetent B. subtilis was prepared and transformed, but the colonies grew poorly on the plates.¹⁹

We attempted to transform pBS1C+Pveg+MFS a second time, more dilutions were plated. The successful colonies were verified via colony PCR and we finally transformed pBS1C + Pveg + MFS into B. subtilis!⁹

The other 5 promoter + backbone + MFS ligations were attempted with mannitol supercompetent B. subtilis.¹⁹The same procedure was followed but only pBS4S + Pveg + MFS worked.

Xylose supercompetent B. subtilis was prepared and the 4 remaining combinations (pBS1C+PliaG, pBS1C+PlepA, pBS4S+PliaG, pBS4S+Plep) were transformed.²⁰ Only pBS1C + PlepA + M grew, and the colonies were verified to be successful using colony PCR.⁹

A second and final attempt at transforming the remaining 3 promoter+backbone+MFS was made. However, the colony PCR showed no successful transformation.⁹

Following a CFU to OD600 standardization, a failed cholesterol uptake assay using colorimetric quantification was performed.²⁶

Another modified cholesterol uptake assay was attempted, giving good standards. However, the bacteria interfered with the colorimetric assay, causing inaccurate results.²⁶

Fluorometric cholesterol uptake assay was attempted.²⁶Although the standard deviations were large, B. subtilis with MFS clearly uptook more cholesterol than the E. coli control.

To create our biobrick, pJUMP28 and Phag were ligated and transformed into NEB DH10b.²⁷

More replicates for the fluorometric cholesterol assay were attempted to lower the standard deviation.²⁶ B. subtilis with MFS appeared to make no significant difference in cholesterol uptake, but wild-type B. subtilis uptakes more than E. coli.^3,9

Gibson assembly was performed to create our biobrick pBSChag, an integrative vector that integrates into flgM site in B. subtilis that encodes for flagellum genes.^24,31 Backbone and inserts fragments were PCR amplified and purified, DpnI restriction digest verified successful amplification for Gibson assembly.^4,11

pBSChag Gibson product was transformed into BL21 twice unsuccessfully.¹² Endura electrocompetent E. coli was used for transformation, which worked. Colony PCR confirmed successful transformation.⁹

Sequencing results for pBSChag Gibson product confirmed successful Gibson assembly with no mutations.

Phag was cloned into pBSChag via restriction digestion with EcoRI and PstI, ligated with T4 DNA ligase, then transformed into OneShot TOP10 cells.^5,8 Successful colonies were verified via diagnostic restriction digestion with EcoRI and PstI.⁵

No experiments were run in this project portion this week.

No experiments were run in this project portion this week.