• glpX
• We tried to find and isolate pSB1K3_glpX.
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~1300 bp)
• Plasmid isolation of pSB1K3_glpX

• csoS1-4
• We tried to amplify csoS1-4 (shell proteins) for a fusion with GFP.
• PCR amplification (fw-GFP-csoS1B, rv-rv-GFP-csoS1A)
• Annealing temperature: 54 °C
• Bands as expected (~4000 bp (~1800 bp csoS1-4, ~2200 bp pSB1C3))
• PCR products were purified

• csoS1-4 and GFP (BBa_K1465222)
• We tried to assemble the shell proteins of the carboxysome and GFP.
• Gibson Assembly with pSB1C3_csoS1-4 and GFP
• Restriction digestion with DpnI
• Transformation with electrocompotetent cells
• Colony PCR (fw-csoS1A-GFP, rv-csoS1B-GF)
• Annealing temperature: 68 °C
• Bands as expected (~1000 bp)
• Plasmid isolation of pSB1C3_csoS1-4_GFP
• Restriction digestion with NotI
• Bands as expected (~2000 bp and ~2400 bp)

Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

• csoS1-4_GFP and T7
• We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promotor.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1A2_T7
• Insert (digested with XbaI, PstI)
• csoS1-4_GFP
• Transformation with electrocompotetent cells
• Colony PCR (VF-Primer, rv_csoS4A_PstI)
• Annealing temperature: 55 °C
• Bands as expected (~320 bp)

• can and csoS1-4 (BBa_K1465208)
• This week we tried to find positive clones of our transformation.
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~3600 bp)
• Plasmid isolation of pSB1C3_can_csoS1-4
• Restriction digestion with EcoRI and PstI
• Bands as expected (~2100 bp and ~3300 bp)

Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

• can_csoS1-4 and csoS1D (BBa_K1465209)
• We tried to assemble our pSB1C3_can_csoS1-4 construct with csoS1D.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1C3_can_csoS1-4
• Insert (digested with XbaI, PstI)
• csoS1D
• Transformation with electrocompotetent cells
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~4300 bp)
• Plasmid isolation of pSB1C3_can_csoS1-4_csoS1D
• Restriction digestion with NotI
• Bands as expected (~2000 bp and ~4000 bp)

Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

• Hneap and T7
• This week we tried to assemble the T7 promotor with the Hneap.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1A2_T7
• Insert (digested with XbaI, PstI)
• Hneap
• Transformation with chemocompetent cells

• glpX and p_tac (BBa_K1465229)
• This week we tried to bring glpX in the pSB1C3 backbone under the control of the p_tac promotor.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1C3_p_tac
• Insert (digested with XbaI, PstI)
• glpX
• Transformation with electrocompotetent cells
• Colony PCR (fw_pSB1_SBPase, rv_pSB1_SBPase)
• Annealing temperature: 54 °C
• Bands as expected (~2100 bp)
• Plasmid isolation of pSB1C3_p_tac_glpX
• Restriction digestion with NotI
• Bands as expected (~2000 bp and ~2200 bp)


• Purification of the carboxysome
• Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling.
→ The purification was not succesful, as you could not recognize a visible band in the gradient.

• Hneap and T7
• We tried to find positiv clones of pSB3A2_T7_Hneap.
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~2000 bp)
• Plasmid isolation of pSB1A2_T7_Hneap
• Restriction digestion with EcoRI and PstI
• Bands as expected (~2000 bp and ~1800 bp)

Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

• sap (BBa_K1465204)
• We tried to assemble both parts of sap.
• PCR amplification on the pJet plasmid for sap_2 (sap_2_fwd, sap_2_rev)
• Annealing temperature: 55 °C
• Bands as expected (~1500 bp)
• Gibson Assembly with sap_1, sap_2 and pSB1C3
• Transformation with electrocompotetent cells
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~3000 bp)
• Restriction digestion with EcoRI and PstI
• Bands as expected (~2000 bp (backbone) and ~2700 bp (insert))
• Restriction digestion with EcoRV
• Bands as expected (~1500 bp and ~3200 bp)

Agarose gel from restriction digestion. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

• can_csoS1-4 respectively can_csoS1-4_csoS1D and sap
• We tried to finish our carboxysome with and without csoS1D but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.


• Purification of the carboxysome
• Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling.
→ The purification was not succesful, as you could not recognize a visible band in the gradient.

• T7 and prkA
• This week we wanted to bring the prkA under the control of the T7 promotor.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1A2_T7
• Insert (digested with XbaI, PstI)
• prkA
• Transformation with chemocompetent cells
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~1400 bp)
• Plasmid isolation of pSB1A2_T7_prkA
• Restriction digestion with EcoRI and PstI
• Bands as expected (~2000 bp and ~1100 bp)

• T7 and sap
• We tried to bring the sap under the control of the T7 promotor.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1A2_T7
• Insert (digested with XbaI, PstI)
• sap
• Transformation with chemocompetent cells
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~3000 bp)
• Plasmid isolation of pSB1A2_T7_sap
• Restriction digestion with EcoRI and PstI
• Bands as expected (~2800 bp and ~2000 bp)

• csoS1-4_GFP and T7_sap
• We tried to assemble our GFP construct with the shell associated protein sap and the T7 promotor.
• BioBrick Assembly (Prefix)
• Backbone (digested with EcoRI, XbaI)
• pSB1C3_csoS1-4_GFP
• Insert (digested with EcoRI, SpeI)
• T7_sap
• Transformation with electrocompotetent cells
• Transformation with chemocompetent cells

• T7_sap_csoS1-4_GFP (BBa_K1465223)
• We tried find the construct to use it for the microscopy.
• Colony PCR (VF-Primer, Primer2)
• Annealing temperature: 55 °C
• Bands as expected (~3000 bp)
• Plasmid isolation of pSB1C3_T7_sap_csoS1-4_GFP
• Restriction digestion with PstI and EcoRI
• Bands as expected (~2000 bp and ~4600 bp)

• tkt
• This week we wanted to purify the enzyme of tkt for the SBPase assay.
• Cultivation of pet16b_tkt in 250 ml LB medium
• Induction with 1 mM IPTG at OD 0.8. Taking samples:t₀, t₁, t₂, t₃, t₁₅, t₁₇
• SDS-Page of cultivation result in correct bands at ~100kD
• His-Tag purification of tkt
• SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

• fba
• This week we wanted to purify the enzyme of fba for the SBPase assay.
• Cultivation of pet16b_fba in 250 ml LB medium
• Induction with 1 mM IPTG at OD 0.8. Taking samples:t₀, t₁, t₂, t₃, t₁₅, t₁₇
• SDS-Page of cultivation result in correct bands at ~40kD
• His-Tag purification of fba
• SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

• glpX (BBa_K1465228 (pSB1C3_glpX))
• This week we wanted to purify the enzyme of for the SBPase assay.
• Cultivation of pet16b_glpX in 250 ml LB medium
• Induction with 1 mM IPTG at OD 0.8. Taking samples:t₀, t₁, t₂, t₃, t₁₅, t₁₇
• SDS-Page of cultivation result in correct bands at ~38kD
• His-Tag purification of glpX
• SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...


• We also tried to bring glpX in the right vector pSB1C3.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1C3
• Insert (digested with XbaI, PstI)
• glpX
• Transformation with electrocompotetent cells
• Transformation with chemocompetent cells
• Colony PCR (fw_pSB1_SBPase, rv_pSB1_SBPase)
• Annealing temperature: 54 °C
• Bands as expected (~1300 bp)
• Plasmid isolation of pSB1C3_glpX
• Restriction digestion with PstI and EcoRI
• Bands as expected (~2000 bp and ~1000 bp)


• Additionally we tried to bring glpX under the control of the T7 promotor.
• BioBrick Assembly (Suffix)
• Backbone (digested with SpeI, PstI)
• pSB1A2_T7
• Insert (digested with PstI, XbaI)
• glpX
• Transformation with chemocompetent cells
• Colony PCR (Primer1, Primer2)
• Annealing temperature: ... °C
• Bands (not) as expected (~... bp)

• prkA and RBS BioBrick (BBa_B0034)
• We recognized that our pSB1C_prkA construct did not have the desired RBS so we tried to take the RBS (pSB1A2_RBS) out of the parts distrubution 2014 and clone it in front of our prkA.
• Plasmid isolation of pSB1A2_RBS
• BioBrick Assembly (Suffix/Prefix)
• Backbone (digested with SpeI, PstI)
• pSB1A2_RBS
• Insert (digested with PstI, XbaI)
• prkA
• Transformation with chemocompetent cells
• Colony PCR (VF-Primer, VR-Primer)
• Annealing temperature: 55 °C
• Bands as expected (~1500 bp)
• Plasmid isolation of pSB1A2_prkA
• Restriction digestion with [Enzyme]I and [Enzyme]I
• Bands as expected (~ bp)

• T7_sRNA:pfkA and p_tac_sRNA:pfkA
• We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of E. coli. The cultivations were made in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C. The induction was with rhamnose for the T7 promotor and IPTG for the p_tac. It lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for HPLC analysis that followed.