• 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
• 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)

• 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
• 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)

• can_csoS1-4 and csoS1D
• 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)

• 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 electrocompotetent cells

• 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)

• sap
• 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)