Protocols

Weekly lab work

May

May 1-5

Our first week in the lab consisted of revivification and the beginning of the cultivation process of our received C.crecestus and H. baltica strains. We also conducted a full inventorization of our laboratory.

May 6-12

This week started with the ending of the bacteria cultivation process. After that, we purified their genomic DNA from C. crescentus CB2, CB2A strains, and H. baltica, which we will use in the future. Also, a batch of electrocompetent E. coli Mach1cells was made, and their efficiency was tested. First, we attempted to amplify our genes using PCR (polymerase chain reaction).

Preparing stock solutions for our gene knockout experiments. Making the necessary LB agar plates for the arriving pCas9 and pCRISPR-SacB plasmids. Preparing electrocompetent KRX strain cells.

May 13-19

We continued our work of amplification. On the side, stocks of antibiotics were made, and we will use them to grow overnight mini preps and LB agar plates.

Plated received plasmids (pCasRed plasmid - pCas9, pCRISPR-SacB plasmid - pCRISPR, pCas9, and pTargetF (the last two received from Songailienė)).

May 20-26

The week started with the last PCR reactions being finished. During this time, we prepared our vectors (pETDuet and pACYCDuet) for the GG (Golden Gate) reaction. By the end of the week, the first attempts of GG reactions were made, and the products were electroporated into Mach1 electrocompetent cells.

May 27 - June 2

Along with more gene amplification and GG reactions, we tested the validity of our made constructs with cPCR (colony polymerase chain reaction) alongside RE (restriction endonuclease) reactions. During this week, a new batch of Mach1 electrocompetent cells was tested for their efficiency.

June

June 3-9

This week, the work of assembling our plasmids continued, along with retrying some of the failed GG reactions and PCR. We successfully assembled the CB2 hfsA-hfsB-hfsD plasmid. 

Growing bacteria with plasmids required for gene knock-outs, doing in silico work (primer, homology arm creation), PCR of gDNA fragments, and making necessary stock solutions.

June 10-16

The assembly of plasmids continues. From the already electroporated and grown GG products in our Mach1 strain, we did cPCR (colony polymerase chain reaction), and after plasmid purification, RE reactions were performed. If both tests were successful, we prepared and sent the samples for sequencing. This week we successfully assembled HB hfsF-hfsC-hfsI plasmid.

KLD reaction on gDNA plasmids to make them circular. Transformation of circular plasmids into Mach1 electrocompetent cells. Performed cPCR to check the validity of the plasmid sequence. Did PCR on dDNA for wecA gene knockout. Multiplied pCas9 plasmid.

June 17-23

This week, we kept on assembling and checking our plasmids using the methods mentioned above.

Multiplied pCas9 plasmid and reran dDNA PCRs products through a TBE agarose gel. Purified both the pCas9 plasmids and dDNA PCR products.

June 24-30

The week consisted of plasmid assembly and their sequence validation. This week we successfully assembled CB2 hfsF-hfsC-hfsI plasmid.

Redone dDNA PCR from last week because the wrong primers were used.

July

July 1-7

The lab team split into two groups: half of the team kept at it with plasmid assembly and sequence validation, and the other half started with expression work (multiple tries in KRX strain). So, the job consisted of making overnight cultures and a 6 LB medium flask. After protein expression, the first SDS-PAGE gels were made and run. This week we successfully assembled CB2A hfsA-hfsB-hfsD and CB2A hfsF-hfsC-hfsI.

Performed wecA gene knock-out and prepared gDNAs with dDNAs for other gene targets. Stocked up on gene knock-out solutions. Electrotransformed gDNAs in Mach1 electrocompetent cells. Prepared other required plasmids and sent them for sequencing.

July 8-14

During this week, some significant changes were made. Our plasmid assembly work kept stalling, so we decided to change the vector backbone for our cytoplasmic genes. We were using pETDuet-1 from this week onwards, we used pRSFDuet-1. Also, constructing the plasmids with genes PCR-amplified straight from the genome did not work, so we adjusted and, from this week, used 2-step amplification, meaning first we amplified the genes from the genome and then used the purified PCR product as a matrix for another PCR (overall schematic: Genome -> (first PCR) -> Purified firsts PCR product -> (second PCR, using first PCR’s product as a matrix) -> amplified gene for plasmid assembly). We performed protein expression (KRX strain) and the corresponding SDS-PAGE gels alongside plasmid assembly work. This week we successfully assembled CB2 hfsE-hfsJ-hfsG and CB2 hfsL-hfsH-hfsK. 

Prepared gene knock-out solutions and grew KRX electrocompetent cells (with pCas9 plasmid). Electrotransforming pTargetF plasmid (targeting wecA) and dDNA templates. To validate the sequence PCR was performed.

July 15-21

This week mainly consisted of plasmid assembly and sequence validation. On the side, we made a new batch of electrocompetent Mach1 cells. This week we successfully assembled CB2A hfsL-hfsH-hfsK and HB hfsE-hfsG-hfs. 

Performed experiment to knock out WecF gene. Cured pTargetF and WecA from KRX gene with knocked out wecA gene.

July 22-28

Nothing new happened this week, we kept at it with plasmid assemblies and one protein expression (KRX strain) was performed with SDS-PAGE gel following right after.

July 29 - August 4

The first part of the week consisted of finishing the plasmid assembly part of our project. Of course, not all the plasmids were entirely constructed by this part, but we have the whole system of C. crescentus CB2. There is still some work to be done with the CB2A strain and H. baltica. From this week on, only one lab team member will finish the plasmid assemblies. Also, during this week, the grunt work of protein expression (KRX and BL21 (DE3) strains) started. This week we successfully assembled HB hfsJ-hfsL-hfsK.

August

August 5-11

The lab's primary human resources were focused on protein expressions in different host strains and conditions (BL21(DE3) strain). On the side, all the SDS-PAGE gels are being prepared and photographed. Also, two new batches of electrocompetent cells were made: Mach1 and BL21 (DE3). During all of this, the final plasmid assemblies are finished. This week we successfully assembled HB hfsA-hfsB-hfsD.

August 12-18

This week, we continued protein expression (BL21 (DE3) strain) work. Our lab lead tried to purify our final product holdfast (the protocol was adapted from several other protocols; you can see the final version in the protocol book). The product from the purification experiment was used for our first WGA blot (the protocol is also in the protocol book). During this time, work for ori/antibiotic-changed plasmids started. We finished the week by preparing our samples and going to ThermoFisher Scientific for proteomics. This week we successfully assembled CB2A hfsE-hfsJ-hfsG.

Made overnight cultures for freshly transformed wecA gDNA constructs. We decided to change our strategy for gene knockouts, and created new primers for the required constructs.

August 19-25

We are continuing the work started a week ago for the antibiotic switch. Expression (BL21 (DE3) and C41 (DE3) strains) work is still continuing. We are narrowing down the optimal conditions and determining what host works best. Some of the best results harboring expressions were repeated on a larger scale for purification experiments.

Redone gene knock-out. Made electrocompetent BL21 (DE3) cells with pCas9 plasmid and purified new dDNA. Made overnight cultures of BL21 (DE3) + pRED and p708 (flipase) plasmids, also HMS174 + pCas9.

August 26 - September 1

During this intense week, a couple of expressions (BL21 (DE3) and C41 (DE3) strains) were performed and left overnight to form rings with glucose. All the lab's human resources were poured into amplifying and running GG reactions with our chosen genes, which will be purified using 6X histidine markers and spin columns. Before the protein purification process started, we checked the validity of every plasmid using already discussed methods. Also, another dot blot was performed. We plated our ring formations onto various plates (the plates varied in IPTG concentration and antibiotics, for in depth information see “Engineering”). The week ended with the first attempts at holdfast purification.

Purified pRED and p708 plasmids. Made electrocompetent cells (BL21 (DE3), DH10B, C41 (DE3), Rosetta(DE3)pLysS). Made HMS174 (DE3) electrocompetent cells and electrotransforming pRED plasmid into them.

September

September 2-8

Starting this week with a new batch of Mach1 electrocompetent cells. Expression work started again (Rosetta, BL21 (DE3) and C41 (DE3) strains). Another dot blot was prepared with the products obtained from last week's holdfast purification experiment. We expressed all of our single gene plasmids that were prepared last week. Bioreactor tests were executed.

Performed homology recombineering gene knock-out for DH10B and HMS174(DE3). PCR’ed the products and checked them on 1% TAE agarose gels.

September 9-15

The last of our expressions (BL21 (DE3) strain) were performed, and minor finishing work. Also, a WGA dot blot assay was performed.

Performed homology recombineering gene knock-out of wecA and wecB genes for E. coli strains DH10B, HMS174(DE3), BL21(DE3), Rosetta(DE3)pLysS, C41(DE3). PCR’ed the products and checked them on 1% TAE agarose gels.

September 16-22

Expressions (BL21 (DE3) and HMS174 (DE3) strains) and WGA blot were performed. A bioreactor experiment was performed. We closed the week off with a deep clean of the lab. SEM experiments were done with the help of colleagues from the Faculty of Chemistry and Geosciences.

September 23-29

This week we tested our bioreactor. We did FTIR and bright field microscopy to analyze holdfast-producing bacteria. Knocked-out strains were tested for protein and holdfast production.