Team:Bielefeld-CeBiTec/Notebook/HeavyMetals

iGEM Bielefeld 2015


Heavy metals Date rape drugs CFPS PRIA Miscellaneous

Heavy Metals



Week 1 (4/20/2015-4/26/2015)

  • We transformed BioBricks from the distribution:
    • BBa_J33201 (arsR)
    • BBa_K516030 (mRFP1 with RBS and terminator)
    • Colonies were used to inoculate overnight cultures.
    • Plasmids were isolated using a miniprep kit.

Week 2 (4/27/2015-5/3/2015)

  • We transformed BioBricks from the distribution:

Week 3 (5/4/2015-5/10/2015)

  • ArsR (BBa_J33201) and RFP (BBa_K516030) were combined using 3A assembly:
    • Restriction digest of arsR, RFP and pSB1K3.m1
    • PCR Clean-up
    • Ligation: 2 µL per fragment, 30 min
    • Transformation by electroporation and heat shock (for comparison)
    • No colonies, therefore the ligation was repeated with significantly more DNA. Incubation overnight.
    • Electroporation resulted in colonies, which were screened with a colony PCR.
    • A product length of 1705 bp was expected. The gel shows that most plasmids seem to be correct.
    • This result was confirmed by sequencing.
  • We repeated the Transformation of BioBricks from the distribution:
  • Gibson-Primerdesign

Week 4 (5/11/2015-5/17/2015)

...

Week 5 (5/18/2015-5/24/2015)

  • Amplification of rcnR and cueR for Gibson Assembly with Taq-Polymerase used E. coli K12 as template
    • rcnR amplification at 77.0°C and 78.9°C with rcnR.rev/fwd primes
      • PCR worked, positive control worked, no amplification of rcnR.
    • cueR amplification at 71.9°C and 74.6°C with cueR.rev/fwd primers
      • PCR worked, positive control worked, no amplification of cueR.
  • Amplification of rcnR and cueR for Gibson Assembly with Phusion Polymerase used E. coli K12 as template
    • rcnR amplification at 54.8 °C and 56.1°C with rcnR.rev/fwd primers
      • PCR worked, positive control worked, no amplification of rcnR.
    • cueR amplification at 61.7°C and 62.8°C with cueR.rev/fwd primers
      • PCR worked, positive control worked, no amplification of rcnR.

Week 6 (5/25/2015-5/31/2015)

...

Week 7 (6/1/2015-6/7/2015)

  • rcnR amplification out of E. coli K12
    • phuison PCR with temerature gradient between 55-63°C
      • with pSB1C3_rcnR_fwd/rev (calulated annealing temperatures 66°C and 71°C
      • with pSB1C3_copAP_fwd/rev (calulated annealing temperatures 53°C and 73°C
        • PCR worked, positive control worked, no amplification of copAP and rcnR.
  • CueR amplification out of E. coli K12
    • phuison PCR with temerature gradient between 55.6-67.6°C
      • with pSB1C3_rcnR_fwd/rev (calulated annealing temperatures 66°C and 71°C
      • with pSB1C3_copAP_fwd/rev(calulated annealing temperatures 53°C and 73°C
        • PCR worked, positive control worked, no amplification of copAP and rcnR.

Week 8 (6/8/2015-6/14/2015)

  • Insertion of a second repressor binding site into BBa_J33201
    • Two primer pairs (Cm_fwd, Ars_1_rev, Ars_2_fwd, Cm_rev) were used to amplify the plasmid and introduce a second repressor binding site after arsR
    • A PCR with Phusion Polymerase failed. A gradient PCR did not show any improvement.
    • A comparison of Phusion and Q5 showed that Q5 works very well.
    • However, an upscalling of the reaction to produce more product did not work.
    • Therefore, two 20 µL reactions were used per fragment. This PCR worked well and the products were extracted from the gel.
    • We performed a Gibson assembly reaction with the two fragments and transformed the mix via heat shock.
    • A colony PCR was performed with five colonies, but only one reaction yielded product.
    • Five colonies were used to inoculate overnight cultures.
    • Plasmids were isolated from the cultures and a restriction digest was performed to check them.
    • The bands were as expected for all colonies.
    • One plasmid was handed in for sequencing, which confirmed its correctness.
  • Gerneration of Gibson fragments
    • phuison PCR with temerature gradient between 55.6-67.6°C
  • CueR amplification out of E. coli K12
    • KOD Polymerase PCR of CueR at 60.4°C, 61°C, 61.7°C and 62.3°C
        • PCR worked, positive control worked, no amplification of cueR.
  • rcnR amplification out of E. coli K12
    • KOD Polymerase PCR of rcnR at 60.4°C, 61°C, 61.7°C and 62.3°C
        • PCR worked, positive control worked, no amplification of rcnR.
  • Gerneration of Gibson fragments

Week 9 (6/15/2015-6/21/2015)

  • 3A assembly of the modified arsR and RFP (BBa_K516030)
    • Restriction digest of the modified arsR with EcoRI-HF and SpeI-HF
    • Heat inactivation: 20 min at 80 °C
    • Ligation with RFP and pSB1K3 (from week 3)
    • Electroporation with 2 µL ligation mix
    • Only one colony grew on the plates, which according to a colony PCR was not correct.
    • 5 and 20 µL of the ligation mix were transformed again into chemocompetent cells.
    • This time there were plenty of colonies. 10 were screened with a colony PCR and most appeared to be correct.

Week 10 (6/22/2015-6/28/2015)

  • First test with arsenic sensor
    • 5 ml cultures (LB + kan) were prepared from glycerol stocks of E. coli harboring pSB1K3-arsR-RFP and pSB1K3-arsR-arsO-RFP and cultured at 37 °C.
    • After 16 h, cultures were diluted 1:100 with fresh LB + kan.
    • After 1 h at 37 °C, arsenic at 1000 µg/L was added.
    • After 24 h at 37 °C, the cultures were centrifuged (5 min, 5000xg).
    • The cell pellets were clearly red, indicating that the arsenic sensors are functional.
      • Cell pellets

Week 11 (6/29/2015-7/5/2015)

  • Arsenic sensor test in 96 well plate
    • Dilutions of a 50 mM sodium arsenite solution were prepared
    • As for the first test, 5 mL cultures of the two strains were inoculated from glycerol stocks.
    • After 18 h, the cultures were diluted to reach an OD600 of 0.4.
    • After 30 min at 37 °C, 100 µL culture and 100 µL sodium arsenite solution were combined in a black 96 well plate.
    • The final arsenic concentrations ranged from 0 to 1000 µg/L.
    • Every combination of strain and concentration was measured in triplicate.
    • The plate was incubated at 37 °C for 4 h.
    • RFP fluorescence was measured in a plate reader.
    • No increase of fluorescence with the arsenic concentration was observable.
    • Measuring different dilutions of an RFP culture supernatant showed that the linear range was very small. Therefore, this instrument does not seem to be suitable for RFP measurements.
  • Repitition of the Phusion PCR amplification for Gibson assembly from week 10 using Primermixes
    • Cm_fwd + rcnA_rev (expected Primerlength:838 bp)
    • Cm_fwd + copAP_rev (expected Primerlength:837 bp)
    • Cm_fwd + Cr_rev (expected Primerlength:834 bp)
    • Cm_fwd + pbrA_rev (expected Primerlength:832 bp)
      • Amplification of the fragments was successfull.
      • Following gelextraction of the samples Cm_fwd + rcnA_rev, Cm_fwd + copAP_rev, Cm_fwd + Cr_rev, Cm_fwd + pbrA_rev
  • Q5 PCR amplification of pSB1C3 backbone for Gibson Assembly with cueR and rcnR overlaps used split primers

Week 12 (7/6/2015-7/12/2015)

  • Optimization of arsenic sensor for CFPS
    • We decided to modify the arsenic sensor so that it is likely to work better in the CFPS.
    • The devices we decided to clone are:
      • An sfGFP under the control of the T7 promoter and the arsenic operator including the UTR which, as we showed, improves the sfGFP expression in the CFPS.
      • An sfGFP with the UTR behind arsR and its natural promoter.
      • A his-tagged arsR for purification and controlled addition to the CFPS reaction.
    • In the first step, the T7 promoter needed to be inserterd in front of arsR. Therefore, BBa_I719005 (T7 promoter) from the distribution was transformed by heat shock.
    • A colony PCR showed bands which matched the expected length.
    • Overnight cultures were inoculated with the colonies and plasmids were isolated.
    • Restriction digest:
      • pSB1A2-pT7 with EcoRI-HF and SpeI
      • pSB1K3-arsR-RFP with XbaI and PstI
      • pSB1C3 (linearized) with EcoRI-HF, PstI and DpnI (+ FastAP)
    • Ligation of the tree fragments and heat shock transformation resulted in small colonies, which were not correct according to a Colony PCR.

Week 13 (7/13/2015-7/19/2015)

  • Optimization of arsenic sensor for CFPS
    • The ligation of T7 promoter, arsR and pSB1C3 was repeated, and this time there were several colonies which showed the expected band after a colony PCR.
    • Several PCRs were performed to create fragments for Gibson Assembly:
      1. Ars_3_fwd and Cm_rev on BBa_J33201
      2. Cm_fwd and Ars_3_rev on BBa_J33201
      3. Cm_fwd and UTR_noT7_rev on BBa_I746909
      4. UTR_fwd and Cm_rev on BBa_I746909
    • All reactions worked very well and the products were extracted from the gel.
    • The products from PCR 1+2 and 3+4 were combined by Gibson Assembly.
    • Following heat shock transformation, plenty of colonies grew on the plates.
    • Some were checked by Colony PCR and all seemed to be correct.
    • 3 overnight cultures per construct were inoculated and plasmids were isolated.
    • The isolated plasmids were used for further cloning steps:
    • A restriction digest was performed with the isolated pSB1C3-UTR-sfGFP (XbaI and PstI) and pSB1K3 (EcoRI-HF, PstI, DpnI, FastAP)
    • Ligation:
      • arsR-arsO2 + RFP + pSB1K3
      • arsR + UTR-sfGFP + pSB1K3
    • PCR:
    • The products were extracted from the gel and combined via Gibson Assembly.
    • The Gibson Assembly and ligation mixes were transformed via heat shock.
  • Q5 PCR amplification of pSB1C3 backbone with cueR overlaps using split primers at 63°C
  • Phusion PCR amplification of pSB1C3 backbone with konstitutive promoter and pSB1C3 backbone with pbrR overlaps using split primers
    • Cm_rev + kProm_fwd (expected Primerlength:1320 bp) at 63°C
      • Amplification of the Cm_rev + kProm_fwd was not successfull.
    • Cm_fwd + pbrR_rev (expected Primerlength:835 bp) 68°C
      • Amplification of the Cm_fwd + pbrR_rev was successfull.
    • Following gelextraction of the samples Cm_rev + pSB1C3_Cuer_suf and m_fwd + pSB1C3_cueR_pre
  • Phusion PCR amplification with temperature gradient between 62°C-70°C:
    • Cm_rev + RFP_fwd at 63°C
    • Cm_fwd + pSB1C3_cueR_suf at 68°C
    • Cm_rev + kProm_fwd at 63°C
      • PCR worked, positive control worked, successfull amplification of pSB1C3-suf_RFP, pSB1C3-suf_CueR and pSB1C3-suf_kProm.
      • Following gelextraction of the samples Cm_rev + pSB1C3_Cuer_suf and m_fwd + pSB1C3_cueR_pre
  • Gibson Assembly with former generated fragments:
    • for assembly used both split primer backbone fragments with overlap and the synthesized promoter + RFP or constitutive promoter + specific activator/ repressor
    • transformation into E. coli via heatshock
  • Guideline for Induction levels Trinkwasserschutzverordnung and WHO guidelines
    • no growth inhibition

Week 14 (7/20/2015-7/26/2015)

  • Optimization of arsenic sensor for CFPS
    • The colonies which had grown after Gibson assembly and transformation were checked with a colony PCR. The bands were as expected.
    • Overnight cultures were inoculted and the plasmids were isolated and handed in for sequencing.
    • The sequencing results for arsR-UTR-sfGFP were slighly unclear at one position, but likely the device is correct.
    • All other devices were clearly correct.
  • Colony PCR with Taq Polymerase of Gibson clones(Gibson Assembly from week 13) using VF and VR Primers:
    • Template: some bacteria resuspended in 50µL steril water and lysed by heat at 90°C for 10min
    • potential right clones were transferred to new LB agar plates with Chlorampohenicol
      • CrProm
      • copAP
      • CueR
      • rcnA
      • pbrA
      • pbrR
    • Plasmid DNA isolation using thermo fisher miniprep kit and analytic Jena kit
    • The colonies which had grown after Gibson assembly and transformation were checked with a colony PCR with Taq-Polymerase:
      • rcnR
      • rcnA
      • merR
      • chrB
      • pbrA
      • potential right clones (rcnA, merR, pbrA, rcnR) were transferred to new LB agar plates with Chlorampohenicol, Plasmid DNA was isolated with thermo fisher miniprep kit and handed in for sequencing
  • Repetition of growth inhibition test with higher heavy metal concentrations
    • lead: 2 g/L and 20g/L
    • mercury: 6 g/L and 60 g/L
    • chromium: 6 g/L and 60 g/L
    • nickel: 8 g/L and 80 g/L
    • copper: 20 g/L and 40 g/L
  • KOD Amplification of Gibsonfragments for merR and rcnR with temperature gradient between 64°C-68°C, sfGFP and RFP used as template
    • no desired fragments
  • KOD Amplification of Gibsonfragments for merR and rcnR with temperature gradient between 52°C-54°C, sfGFP and RFP used as template
    • The bands were as expected.
    • PCR clean-up of rcnR using M&N PCR clean-up and gel extraction kit
    • Gel extraction of merR using M&N PCR clean-up and gel extraction kit
  • Repetition of PCR for kProm fragment and RFP backbone fragment
    • afterwards PCR cleanup
  • Gibson Assamby for MerR and RcnR
    • afterwards heat shock transformation
      • The colonies which had grown after Gibson assembly and transformation were checked with a colony PCR. The bands of one merR was as expected.

Week 15 (7/27/2015-8/2/2015)

  • Cloning of a his-tagged arsenic repressor
    • To get an arsenic repressor which can be purified and added to the CFPS, a his-tagged version under the control of the T7 promoter was cloned.
    • The first step were the following PCRs:
    • The products were extracted from the gel.
    • Gibson Assembly and heat shock transformation were performed.
    • A colony PCR indicated that the cloning was successful, which was confirmed by sequencing.
  • Colony PCR of RcnR clones
    • colony PCR indicated that the Gibson assembly was not successful
  • Biobrick assembly to generate a plasmid out of cueR and copAP
    • Restriction of konst. Prom. + cueR BBa_K1758320 with EcoRI and SpeI
    • Restriction of CopAP_RFP BBa_K1758321 with EcoRI and XbaI
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was not successful
  • Biobrick assembly to generate a plasmid out of pbrA and pbrR
    • Restriction of konst. Prom. + pbrR BBa_K1758330 with EcoRI and SpeI
    • Restriction of pbrA + mRFP BBa_K1758331 with EcoRI and XbaI
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was not successful
  • Generation of kProm + rcnR BBa_K1758350 and rcnA +mRFP mRFP BBa_K1758351
    • Gibson assembly of rcnR- and rcnA-fragments
      • heat shock transformation
        • colony PCR of rcnR and rcnA clones
          • colony PCR indicated that the Gibson assembly of rcnR was not successful and rcnA was successful
    • KOD amplification of rcnR backbone with overlaps at 54°C
      • gel extraction of amplified rcnR backbone
  • 3A assembly to generate a plasmid out of pbrA and pbrR
    • Restriction of konst. Prom. + pbrR BBa_K1758330 with EcoRI and SpeI
    • Restriction of pbrA + mRFP BBa_K1758331 with PstI and XbaI
    • ligation of former restrictions
      • colony PCR of ligation
        • colony PCR indicated that the ligation was not successful
  • 3A assembly to generate a plasmid out of cueR and copAP
    • Restriction of konst. Prom. + cueR BBa_K1758320 with EcoRI and SpeI
    • Restriction of CopAP_RFP BBa_K1758321 with PstI and XbaI
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was not successful
  • 3A assembly to generate a plasmid out of chrB and crProm
    • Restriction of konst. Prom. + chrB BBa_K1758310 with EcoRI and SpeI
    • Restriction of Chromium-Prom. + RFP BBa_K1758340 with PstI and XbaI
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was not successful

Week 16 (8/3/2015-8/9/2015)

  • Expression of arsenic repressor
    • An overnight culture was inoculated from the glycerol stock which had been prepared the previous week.
    • Two 200 mL cultures in 1 L shaking flasks were inoculated to an OD600 of 0.1. and cultivated at 37 °C and 180 rpm.
    • At an OD of 0.6, expression of the T7 polymerase was induced by adding rhamnose to a final concentration of 0.1 %.
    • The cultures were grown overnight at 18 °C and 130 rpm.
    • After 24 h, the bacteria were harvested by centrifugation (10 min, 5500xg).
    • The cells were lysed by sonification.
    • Purifiaction was carried out with the Macherey-Nagel Protino Ni-TED 1000 Packed Columns kit.
    • An SDS-PAGE was done to analyze the purification.
  • Measurement of the arsenic sensors´ background signal
    • To reduce the background signal of the arsenic sensor, a second operator site had been introduced downstream of arsR. Due to different information concerning the exact position of the operator, two versions had been prepared.
    • To find out if this had an effect, the following devices were tested:
      • pSB1C3-arsR
      • pSB1K3-arsR-RFP
      • pSB1K3-arsR-arsO1-RFP (first version of the second operator)
      • pSB1K3-arsR-arsO2-RFP (second version of the second operator)
    • Single colonies were obtained from the glycerol stocks by plating.
    • Five cultures per device were inoculated with single colonies.
    • After 15 h, the RFP fluorescence and the OD600 were measured with a Tecan Infinite M200.
    • The RFU was normalized by diving it by the OD and subtracting the singal of plain LB medium.
    • The results showed that the modifications had not reduced the background signal. Instead, it was even higher than that of the unmodified device.
      • Background fluorescence of arsenic devices
  • 3A assembly to generate a plasmid out of pbrA and pbrR
    • Restriction of konst. Prom. + pbrR BBa_K1758330
      • gel extraction with M&N gel extraction kit
    • Restriction of pbrA + mRFP BBa_K1758331 with PstI and XbaI
    • gel extraction with M&N gel extraction kit
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was not successful
  • 3A assembly to generate a plasmid out of cueR and copAP
    • Restriction of konst. Prom. + cueR BBa_K1758320 with EcoRI and SpeI
      • gel extraction with M&N gel extraction kit
    • Restriction of CopAP_RFP BBa_K1758321 with PstI and XbaI
      • gel extraction with M&N gel extraction kit
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was successful, generation of CueR + CopAP + mRFP BBa_K1758322
  • 3A assembly to generate a plasmid out of chrB and crProm
    • Restriction of konst. Prom. + chrB BBa_K1758310 with EcoRI and SpeI
    • Restriction of Chromium-Prom. + RFP BBa_K1758340 with PstI and XbaI
    • ligation of former restrictions
    • heat shock transformation
    • colony PCR of ligation
      • colony PCR indicated that the ligation was not successful
  • PCR amplification of pSB1K3 with KOD
    • gel extraction of the fragment for 3 A assembly
  • Gibson assembly of rcnR (out of E.coli K12) rcnR and rcnA with syntesised rcnA
    • colony PCR indicated that the Gibson assembly of rcnR was successful
    • colony PCR indicated that the Gibson assembly of rcnA was successful
      • This result was confirmed by sequencing.
  • Gibson assembly of konst. Prom. + chrB BBa_K1758310, with syntesised chrB
    • heat shock transformation
    • colony PCR indicated that the Gibson assembly of chrB was not successful

Week 17 (8/10/2015-8/16/2015)

  • Colony PCR of multiple Transformations of Lead Ligation and konst. Prom. + chrB BBa_K1758310, Gibson Assembly
  • Gibson-Assembly of konst. Prom. + chrB BBa_K1758310 (with synthesized chrB):
    • Amplifikation of ChrB overlap part for Gibson-Assembly.
    • RcnR Amplifikation for removing the PstI restriktionsite with Mutagenesis primer, used E.coli K12 as template.

Week 18 (8/17/2015-8/23/2015)

  • Optimization of arsenic repressor for CFPS
    • In order to thest whether it is possible to build a CFPS biosensor by co-expressing the repressor protein in the reaction, the his-tagged arsenic repressor plasmid was optimized.
    • A double terminator was inserted by 3A assembly. However, a test digestion showed unexpected bands.
    • Sequencing confirmed that the double terminator was in fact a different BioBrick.
  • Assembly of sfGFP with promoter (change RFP with UTR+sfGFP)
    • PCR-amplification of copAP, CrProm, pbrA and rcnA with KOD
      • Gel extraction of generated DNA-fragments
    • Gibson assembly of sfGFP with promoters
      • heat shock Transformation of Gibson-Assembly of copAP, CrProm, pbrA, rcnA
      • colony PCR
      • colony PCR indicated that all gibsons (copAP, CrProm, pbrA and rcnA) were successful
      • Sequencing confirmed that the UTR and sfGFP was inserted in all constructs.
  • Construction of konst. Prom. + chrB BBa_K1758310
    • Amplifikation of ChrB overlap part for Gibson-Assembly used chrB_fwd+ Cm_rev
      • Gibson Assembly of ChrB
        • heat shock transformation
        • colony PCR
        • colony PCR indicated that all gibson chrB was successful
        • Sequencing confirmed the result.
    • Repetition of rcnR Gibson Assembly
      • Amplification of RcnR with KOD polymerase
      • PCR cleanup of KOD PCR of 17.8 using M&N cleanup kit
      • Gibson Assembly
      • heat shock transformation
      • colony PCR
      • colony PCR indicated that all gibson rcnR was successful
      • Sequencing confirmed the result.
    • RcnR Amplifikation for removing the PstI restriktionsite with Mutagenesis primer
      • PCR clean up with M&N PCR clean up kit
      • Restruction of rcnR to check the success of the PCR
  • Creation of the merT-5-UTR-sfGFP BBa_K1758342 construct:
    • Amplification of sfGFP with merT overlap used primermix of UTR-merT_fwd and Cm_rev (calculated annealing temperature 56°C and 59°C)
        • Amplification of sfGFP with merT overlap was successful
    • Amplification of pSB1C3 backbone with merT overlap used primermix of pSB1C3-merT_rev and Cm_fwd (calculated annealing temperature 67°C and 63°C)
      • Amplification pSB1C3 backbone with merT overlap was successful
    • PCR cleanup of KOD PCR of 17.8 using M&N cleanup kit
    • Gibson Assembly
    • Heat shock transformation
    • Colony PCR of merT clones
    • Colony PCR indicated that the Gibson assembly of merT was successful

Week 19 (8/24/2015-8/30/2015)

  • Optimization of arsenic repressor for CFPS
    • The UTR was inserted between the T7 promoter and arsR-his for expression in a CFPS reaction.
    • A PCR was performed with BBa_K1758301 as template and the following primer combinations:
    • The PCR did not work with Q5 and Phusion in HF buffer. However, it worked with Phusion in GC Buffer.
    • The fragments were extracted from the gel, combined by Gibson Assembly and transformed via heat shock.
  • Creation of the biosensor constructs ( kProm + ChrB+ChrP+UTR-sfGFP BBa_K1758313, konst.Prom + CueR+CopAP-UTR-sfGFP BBa_K1758324, kProm+PbrR+PbrA-UTR-sfGFP BBa_K1758333, k Prom + merR+merT-UTR-sfGFP BBa_K1758343 and kProm + rcnR+rcnA-UTR-sfGFP BBa_K1758353 )
    • Restriction of specific promoter –UTR-sfGFP (ChrP-5-UTR-sfGFP BBa_K1758312, CopAP-5-UTR-sfGFP BBa_K1758323,PbrA-5-UTR-sfGFP BBa_K1758332, merT-5-UTR-sfGFP BBa_K1758342 and rcnA-5-UTR-sfGFP BBa_K1758352) with EcoRI and XbaI
    • Restriction of specific constitutive promoter activator/ repressor (konst. Prom. + chrB BBa_K1758310, konst. Prom. + cueR BBa_K1758320, konst. Prom. + pbrR BBa_K1758330, konst. Prom. + merR BBa_K1758340 and kProm + rcnR BBa_K1758350) with EcoRI and SpeI
    • Ligation of associated specific Promoters and activator/ repressor
    • Heat shock transformation of the ligations
    • Colony PCR of ligations
      • Colony PCR indicated that the ligation of konst.Prom + CueR+CopAP-UTR-sfGFP BBa_K1758324, kProm+PbrR+PbrA-UTR-sfGFP BBa_K1758333, k Prom + merR+merT-UTR-sfGFP BBa_K1758343 were successful
      • This result was confirmed by sequencing.
      • Colony PCR indicated that the ligation of ChrP-5-UTR-sfGFP BBa_K1758312 and rcnA-5-UTR-sfGFP BBa_K1758352 were not successful
  • Creation of an T7-promoter localized ahead the specic promoter
    • Restriction of specific promoter –UTR-sfGFP (pbrA-, copAP-, rcnA-, crProm-, merT- UTR-sfGFP) with PstI and XbaI
    • Restriction of T7-Plasmid (pSB1A3) with PstI and SpeI
    • Ligation of specific promoter and T7
    • Heat shock transformation of the ligations
    • Colony PCR of ligations
      • Colony PCR indicated that the ligation of merT+T7 and crProm+T7 were successful
      • This result was confirmed by sequencing.
      • Colony PCR indicated that the ligation of copAP+T7, rcnA+T7 and pbrA+T7 were not successful
  • Characterisation of kProm + merR+merT-UTR-sfGFP BBa_K1758343
    • Cultivation: 50 mL LB-media in 250 mL Erlenmeyer flask at 37°C and 180 rpm (over night culture were inoculated to an o.D. of 0,1)
    • Induction with different Lead-concentrations at o.D. 0,6-0,8
    • Periodic measurement of o.D. and fluorescence (after induction)
  • Characterisation of kProm + pbrR+pbrA-UTR-sfGFP BBa_K1758333
    • Cultivation: 50 mL LB-media in 250 mL Erlenmeyer flask at 37°C and 180 rpm (over night culture were inoculated to an o.D. of 0,1)
    • Induction with different Lead-concentrations at o.D. 0,6-0,8
    • Periodic measurement of o.D. and fluorescence (after induction)
  • Characterisation of kProm + cueR+copAP-UTR-sfGFP BBa_K1758324
    • Cultivation: 50 mL LB-media in 250 mL Erlenmeyer flask at 37°C and 180 rpm (over night culture were inoculated to an o.D. of 0,1)
    • Induction with different Lead-concentrations at o.D. 0,6-0,8
    • Periodic measurement of o.D. and fluorescence (after induction)

Week 20 (8/31/2015-9/6/2015)

  • Characterization of arsenic sensor in vivo
    • Glycerol stocks of pSB1C3-arsR and pSB1K3-arsR-RFP were streaked on plates in order to obtain single colonies.
    • Three overnight cultures per construct were inoculated with single colonies.
    • On the following day, 50 mL cultures in LB with the correct antibiotic were inoculated to an OD600 of 0.1.
    • When the cultures had reached an OD of 0.6-0.7, arsenic solutions were added to the arsR-RFP cultures to final concentrations of 0 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, 500 µg/L and 1000 µg/L.
    • Every 1.5-2 h, the OD600 and the RFP fluorescence were measured in a Tecan Reader.
    • The OD600 of LB medium was subtracted from the other OD values. The RFU was then divided by the corresponding OD and the average of the normalized RFU of the arsR cultures was subtracted from each value.
  • Creation of the biosensor constructs (constitutive promoter repressor/ activator + specific promoter UTR sfGFP)
    • Restriction of specific promoter –UTR-sfGFP (rcnA (BBa_K1758352), crProm (BBa_K175832)) with EcoRI and XbaI
    • Restriction of specific constitutive promoter activator/ repressor (merR (BBa_K1758340), rcnR (BBa_K1758350)) with EcoRI and SpeI
    • Ligation of associated specific Promoters and activator/ repressor
    • Heat shock transformation of the ligations
    • Colony PCR of ligations
    • Colony PCR indicated that the ligation of rcnA + rcnR, and crProm + ChrB were successful
    • This result was confirmed by sequencing.
  • Creation of an T7-promoter localized ahead the specic promoter
    • Restriction of specific promoter –UTR-sfGFP (pbrA-, copAP-, rcnA- UTR-sfGFP) with PstI and XbaI
    • Restriction of T7-Plasmid (pSB1A3) with PstI and SpeI
    • Ligation of specific promoter and T7
    • Heat shock transformation of the ligations
    • Colony PCR of ligations
      • Colony PCR indicated that the ligation of copAP+T7, rcnA+T7 and pbrA+T7 (in pSB1A3) were successful
      • This result was confirmed by sequencing.
  • Characterisation of const.Prom + CueR+CopAP-UTR-sfGFP BBa_K1758324, kProm+PbrR+PbrA-UTR-sfGFP BBa_K1758333, kProm + rcnR+rcnA-UTR-sfGFP BBa_K1758353 and kProm + ChrB+ChrP+UTR-sfGFP BBa_K1758313
    • Measurment of o.D. and fluorescence in BioLector of constructs induced with different heavy metal concentrations

Week 21 (9/7/2015-9/13/2015)

  • Backbone exchange ot specific promoters+T7
    • Restriction of T7-specific promoter –UTR-sfGFP (T7-rcnA, T7-crProm,T7-merT, T7-copAP,T7-pbrA) with EcoRI and PstI
    • Restriction pSB1C3-Vector with EcoRI and PstI
    • Ligation of T7-Promoter-construct + pSB1C3-vector
    • Heat shock transformation of the ligations
    • Colony PCR of ligations
    • This result was confirmed by sequencing.