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 from H. baltica, which we will use in the future. Also, a batch of electrocompetent Mach1 cells was made, and their efficiency was tested. First, we attempted to amplify our genes using PCR (polymerase chain reaction).

May 13-19

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

May 20-26

The week started with finishing the last PCR reactions. 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 electrically competent 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 electrically competent cells and tested their efficiency.

June

June 3-9

This week the work of assembling our plasmids continued, alongside retrying some of the failed GG reactions and PCR.

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.

June 17-23

This week we kept at it with assembling and checking our plasmids with the methods mentioned above.

June 24-30

The week consisted of plasmid assembly and their sequence validation.

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. 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 by Gintarė.

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 and the corresponding SDS PAGE gels alongside plasmid assembly work.

July 15-21

This week mainly consisted of plasmid assembly and sequence validation. On the side, we made a new batch of electrically competent Mach1 cells.

July 22-28

Nothing new happened this week, we kept at it with plasmid assemblies and one protein expression 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 fully constructed by this part, but we have the full 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 started.

August

August 5-11

The lab's primary human resources were focused on protein expressions in different host strains and conditions. On the side, all the SDS PAGE gels are being prepared and photographed. Also, two new batches of electrically competent cells were made: Mach1 and BL21 (DE3). During all of this the final plasmid assemblies are being finished.

August 12-18

This week, we continued protein expression 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 with preparation of our samples which are going to ThermoFisher Scientific for proteomics.

August 19-25

We are continuing the work started a week ago for the antibiotic switch. Expression work is still continuing. We are narrowing down the optimal conditions and trying to figure out what host works best. Some of the best results harboring expressions were repeated on a larger scale for purification experiments.

August 26 - September 1

During this intense week, a couple of expressions 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. The week ended with lab lead's' attempt of holdfast purification.

September

September 2-8

Starting this week with a new batch of Mach1 electrically competent cells. Expression work started again mainly with two host strains: Rosetta and BL21. Another dot blot was prepared with the products obtained from last week's holdfast purification experiment. For the first time, we tried Congo Red coomassie assay and exopolysaccharide formation assay. We expressed all of our single gene plasmids that were prepared last week. Bioreactor tests were executed.

September 9-15

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

September 16-22

One last expression 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

The only work done this week was a test of our bioreactor. We did FTIR and bright field microscopy to analyze holdfast-producing bacteria. Knocked-out strains were tested for protein and holdfast production.