Team:Chalmers-Gothenburg/Notebook

iGEM Chalmers Gothenburg 2020

Notebook, Protocols and Primers


For details about the wet lab procedure, check out the lab notes below! For each week in the lab, methods and results are presented for all our experiments. The method description commonly refers to a standard protocol, all of which are included below as well.

The primers used in the experiments are referred to as numbers in both lab notes and protocols. Under “Primer Excel Sheet”, a summarizing description of the primers and their numbers can be found.

Protocols

Primer Excel Sheet

Week 28
Our first week in the lab! Some preparatory work such as preparing selective media and agar plates, producing competent MG1C655 cells and noting the concentrations of ordered DNA was done. We also proceeded with the promoter characterization from previously and, once the primers arrived, amplified each gene using PCR.

Read PDF »

Week 32
Send positive colonies for sequencing and more screening for this week. Beginning of the week, we tried PCR but we shifted the method to digestion. Since almost all colonies were checked and got result as negative, we tried again from Gibson Assembly as well. They will be screening for next week.

Read PDF »

Week 36
A week consisting of intense digest screening in a last attempt to find positive colonies. We also ran another protein extraction and Western blot, this time with quite good results. Found protein expression for A3 but that they aggregate to a quite large extent.

Read PDF »

Promoter characterization
In this document we describe the laboratory procedure for our contribution, in which we characterized three promoters from the parts registry: BBa_J23104, BBa_J23106 and BBa_J23115. In the notebook, these are referred to as A, B and C, respectively. The process includes introducing each promoter in pSB1C3 upstream of an RFP gene, assembling the plasmid, transforming and cultivating bacteria, and studying the fluorescent levels in fluorescence microscopy and in a plate reader. Details about data analysis are found in the contribution page.

Read PDF »

Week 29
We proceeded by purifying the amplified block segments and started trying to assemble the gene block plasmids through Gibson assembly. Different alternative methods to get better results from the amplification of some of the genes were attempted. As problems arose with the Gibson assembly of the gene blocks, fusion PCR was attempted as an alternative assemble method. The genes were amplified with primers introducing his-tags as a result of the re-design of the project.

Read PDF »

Week 33
More screening, sending positive colonies for sequencing, and tried to measure the concentration of protein. This protein concentration measurement methods will be used for western blot which we will do later of this project.

Read PDF »

Week 37
This week we tried to get protein expression of BphB and decrease the aggregation of BphC by cultivating bacteria to different growth phases before extracting the proteins. Both bacteria were cultivated to exponential and stationary phase respectively and run in western blot.

Read PDF »

Week 30
Problems were identified regarding assembling the block segments and were solved by amplification of the plasmid backbone used in the promoter characterization, as this sequence corresponded to the sequence used when designing the overlaps between the his-tagged block segment and the plasmid backbone for the Gibson assembly. This assembly into a new backbone was attempted for all the his-tagged genes. Further attempts to use fusion PCR to assemble the gene blocks were done.

Read PDF »

Week 34
This week, more screening for positive his-tagged block segment transformants was done, as well as sending the positive colonies for sequencing. We also did our first protein extraction and western blot of three confirmed positive colonies.

Read PDF »

Week 38
No lab done this week, and as a result we have no notes to present.

Week 31
This week, we tried to establish the gene block A plasmid, tried to fix the problem about backbone (pSB1C3), constructed each his tagged strain and screening for them. First, we tried to do normal colony PCR but it had so much noise to check them, we change the methods to extract plasmid before start PCR. We could get some positive colonies so we will send them for sequence for next week and need more screening for other strains.

Read PDF »

Week 35
Further screening of transformants with different his-tagged block segments was the focus of this week. For some of the block segments, new Gibson assembly and transformation were done as all colonies seemed negative. A new protein extraction and Western blot was attempted for all the confirmed positive colonies.

Read PDF »

Week 39
We ran another protein extraction and made another attempt to measure the activity of BphC (A3).

Read PDF »