July

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13.07.2022 - 14.07.2022:

• first start in the lab
• preparation of first media, buffers and reagents

15.07.2022:

• our chemo competitor cells have arrived. We started with a transformation for testing
• creating glycerol stocks from DH5α
• eYFP and mTurq chemical transformation in DH5α

18.07.2022 - 19.07.2022:

• precultured our transformation colonys
• plasmid preapration with 6 colonies from our transformation

19.07. - 20.07.2022:

• PCR amplification of the amplification component pNpuC_CTEV_caged_insert
• 1% gel electrophorese

25.07.2022 - 26.07.2022:

• PCR amplification of the amplification component pNpuN_NTEV_insert
• 1% gel electrophorese
• PCR Clean up

27.07.2022:

• PSB1C3 chemical transformation in DH5α strain (psB1C3 BBa_I20270 & psB1C3 BBa_J364000)

28.07.2022 - 29.07.2022:

• precultivation from PSB1C3 transformation colonys
• plasmid preparation

August

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02.08.2022:

• restriction digest for the amplification component
• digestion of the insert using NpuN and NpuC with restirction enzymes SpeI and PstI
• digestion of the vector PSB1C3 for amplification component
• incubation for 1.5 hours

05.08.2022:

• digestion of the vector PSB1C3 for amplification component
• PCR from eYFP and mTurq

10.08.2022 - 11.08.2022:

• PCR product from eYFP and mTurq (template: PSB1C3) was digested with Dpn1 according to attached protocol
• PCR product from eYFP and mTurq was eluted from the gel

12.08.2022:

• starting gibson assembly with eYFP and mTurq in the vector PSB1C3

23.08.2022 - 25.08.2022:

• after the Gibson assembly a colony PCR from different colonys was performed
• after the colony PCR the product was eluted in a 1% agarose gel

26.08.2022:

• three colonys where picked for protein production (colony 2, 6 and 8)
• precultured colonys 2, 6 and 8 with following plasmid preparation

27.08.2022:

• plasmid preparation from colony 2, 6 and 8
• preparing samples for sequencing the parts

29.08.2022 - 30.08.2022:

• precultured colonys for protein production over night
• first protein production of FRET pair
• testing the fluoreszence via special filters
• second restriction digest for amplification component with differnet enzymes producer

September

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13.09.2022:

• PCR of NpuN and NpuC first PCR was made to add the his-tag at the ORF (long)
• PCR product was eluted in 1% agarose gel

14.09.2022:

• preparing new primers for gibson assembly
• PCR from R_his_Ntev, R_his_Ctev, R_his_Ftev, f_p_TEV_complete_insert, F_his_NFtev, F_his_Ctev

15.09.2022 - 17.09.2022:

• adjust FRET protein production by different factors, such as shaking speed
• for gibson assembly the backbone needs to be first opened
• therefore already FRET-Plasmid (Reporter Component was) purified. It will now be opened by a PCR with two Primers

17.09.2022 - 18.09.2022:

• transformation of the performed assembly
• after the transformation a pre culture was started

19.09.2022:

• plasmid preparation of the intein part (amplification component)
• after the plasmid prep a colony PCR with an gel electrophoreses were done

22.09.2022 - 23.09.2022:

• PCR of FTEV short and FTEV long
• precultured colony from FTEV short and FTEV long
• plasmid preparation and preparation for sequencing

Future plans

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• purification of proteins with HIS-tag
• measurement of the FRET signal on the tecan reader
• testing of the components purified and in cell lysate
• quantification of the test with time progression of the FRET signal