Difference between revisions of "Team:Dundee/Lab Book/Chromate Detector"

Revision as of 16:15, 17 September 2015

K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄

Chromate Biosensor

Lab Book

FluID

FluID is a cell free, all in one spray which will be used to detect and dinstiguish between body fluids found at crime scenes.

Chromate Biosensor

Our chromate biosensor is a device that will be used to detect stainless steel residues left behind on bones in blunt or sharp force trauma cases.

Fingerprint Ageing

This device will search for the presence of a specifc component found in fingerprint deposits whose presence/absence will indicate approximate fingerprint age.

Week Beginning 22/06/2015

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008.

Day 1 - 22/06: Plated BBa_K1058008 from agar stab.

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing. The culture was plated from the agar stab and also a liquid culture set up.

Protocols Used: Plating, Overnight cultures

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Day 2 - 23/06: Purified BBa_K1058008 from liquid culture.

Aim of experiment: To purify BBa_K1058008 for sequence confirmation.

Protocols Used: Miniprep plasmid purification, DNA sequencing

Results Sequencing: Figure 1

Next Steps: PCR of promoter region (pChr) and repressor region (ChrB) of BBa_K1058008 for storage in individual biobricks.

Week Beginning 29/06

Summary

During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.

Day 1 - 29/06: PCR-amplification of pChr and ChrB from BBa_K1058008.

Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.

Protocols Used: PCR, Gel extraction, Restriction Digest

Results: Figure 2

Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Week Beginning 06/07

Summary

During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.

Day 1 - 06/07: Repeated amplification of ChrB. Ligation and transformation of pChr and ChrB.

Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of Escherichia coli. Overnight culture of E. coli DH5-alpha, containing GFPmut2, as a fluorescent reporter for our chromate sensing system

Protocols Used: PCR-amplification, Gel extraction, Restriction digest, Ligation, Overnight cultures

Results: Figure 3

2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw. pChr, ChrBs, and ChrBw were ligated into pSB1C3. pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into E. coli MC1061.

Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3. Ligation of pChr to GFP coding sequence for construction of a reporter plasmid.

Day 2 - 07/07: Purification of GFP and overnight cultures of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw

Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant E. coli MC1061, containing pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw respectively for further size and sequence confirmation. Overnight culture of pUniprom for subsequent ligation of ChrB

Protocols Used: Miniprep, PCR-amplification, Gel extraction, Overnight cultures

Results: Figure 5

Next Steps: Repeat of amplification of ChrB. Sequence and size confirmation of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw.

Day 3 - 08/07: Purification of plasmids pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw. Size and sequence confirmation.

Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.

Protocols Used: Miniprep, PCR amplification, Restriction digest, Sequencing

Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.

Results:

Figure 6 - Size confirmation of pChr and ChrB.

Figure 7 - Sequence confirmation of pChr

Figure 8 - Sequence confirmation of ChrB.

Next Steps: Purification and digest of pUniprom for insertion of ChrB.

Day 4 - 09/07: Digested and cleaned up pUniprom for insertion of ChrB. Amplification of optimised ChrB with primers specific for the insertion into pUniprom.

Aim of experiment: To prepare pUniprom for insertion of ChrB by digesting it with BamHI and HindIII. To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.

Protocols Used:

Restriction digest and Gel extraction of pUniprom

PCR and Gel extraction of optimised ChrB

Results:

Figure 9 Gel with pUniprom

Figure 10 Gel with ChrB

Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.

Day 5 - 10/07: Ligations of ChrB and optimised ChrB into pUniprom, and of pChr and GFPmut2 into pSB1C3.

Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr and GFPmut2 to each other via SpeI/XbaI-link and into pSB1C3 via EcoRI and PstI restriction sites.

Protocols Used: Ligations

Results: N/A

Next Steps: Transformation of abovementioned ligations.

Week Beginning 13/07

Summary

During this week successful transformants were identified and propagated.

Day 1 - 13/07: Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP.

Aim of experiment: Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP into E. coli JM110 chassis. Setting up a backup restriction digest of pSB1C3 with EcoRI and SpeI in case the double ligation pSB1C3-pChr-GFP is unsuccessful.

Protocols Used: Transformations, Restriction digest

Results: Figure 11

Next Steps: Purification of recombinant vectors and confirmation of size and sequence. Store digested pSB1C3 at -20^oC.

Day 2 - 14/07: Set up cultures for subsequent purification of recombinant vectors for size and sequence confirmation.

Aim of experiment: Purification of recombinant vectors, pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP, for size and sequence confirmation. Patch plating of the same colonies for storage and retrieval once size and sequence have been confirmed.

Protocols Used: Overnight cultures, Patch plating

Results: N/A

Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.

Day 3 - 15/07: Purified pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight cultures

Aim of experiment: Purification of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight for confirmation of size and sequence. Restriction digest of pUnipromChrB and optimised pUniprom-ChrB optimised with BamHI and HindIII and of pSB1C3-pChr-GFP with EcoRI/PstI for size confirmation.

Protocols Used: Miniprep, Restriction digest, Colony PCR, Sequencing

Results:

Figure 12 Size confirmation of ChrB and ChrBopt.

Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.

Figure 14 Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.

Next Steps: Confirmation of sequence of ChrB-opt.

Day 4 - 16/07: Set up overnight cultures of colonies chosen after colony-PCR for subsequent purification and sequence confirmation.

Aim of experiment: Set up overnight cultures of ChrBopt colonies 2, 4, 9, and 12 for sequencing.

Protocols Used: Overnight cultures

Results: N/A

Next Steps: Purification of recombinant plasmids.

Day 5 - 17/07: Purified and sequenced chosen ChrBopt-colonies.

Aim of experiment: To purify recombinant plasmids for sequence confirmation.

Protocols Used: Miniprep, Sequencing

Results: ChrBopt showed HindIII restriction sites and was hence cleaved into partial inserts.

Next Steps: Use alternative cloning protocol for ChrB and ChrBopt.

Week Beginning 20/07

Summary

During this week an alternative cloning strategy for ChrB and ChrBopt was employed. Furthermore, the pSB1C3-pChr-GFP construct was cloned in a stepwise fashion.

Day 1 - 20/07: Went back to the original plate of pSB1C3-pChr-GFP for visualising colonies that do or do not express GFP.

Aim of experiment:To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light. The background of this experiment was that if there is one colony that has both inserts in the correct orientation with respect to each other, then there is perhaps one that also has them with the correct orientation with respect to the plasmid. Colonies identified as expressing GFP were patch plated and grown in liquid cultures over night in order to confirm the sequence of the insert.

Protocols Used: Patch-plating, overnight cultures

Results: Figure 15 pSB1C3-pChr-GFP in E. coli JM110.

Next Steps: Sequence confirmation of newly selected colonies.

Day 2 - 21/07: Purified plasmids from new colonies and confirmed sequence. Restriction digest of pUniprom with BamHI and PstI.

Aim of experiment:Purification of newly selected colonies, 1, 5, 6, and 7, containing pSB1C3-pChr-GFP for subsequent sequence confirmation. Restriction digest of pUniprom with BamHI and PstI to employ an alternative cloning strategy of ChrB and ChrBopt.

Protocols Used:

Miniprep and Sequencing of pChrGFP insert.

Restriction digest of pUniprom.

Results:

Figure 16 Alignment of sequencing results.

Figure 17 Gel after restriction digest of pUniprom.

Next Steps:Stepwise ligation of pChr and GFP into pSB1C3 in order to clone the inserts in the correct orientation with respect to the plasmid. Insertion of ChrB and ChrBopt into pUniprom via BamHI and PstI links.

Day 3 - 22/07: Ligated pChr into pSB1C3 via EcoRI and SpeI sites and cloned the recombinant plasmid into E. coli JM110.

Aim of experiment: Ligation of pChr into pSB1C3 via EcoRI SpeI link. This is the first step of a 2-step cloning strategy with the aim of cloning pChr and GFP into pSB1C3 in the correct orientation with respect to the plasmid.

Protocols Used: Ligation, Transformation

Results: Figure 18 Plates after transformation of pChr into pSB1C3.

Next Steps: Sequence confirmation of pChr in pSB1C3.

Day 4 - 23/07: Set up overnight cultures of pSB1C3-pChr colonies.

Aim of experiment: Set up overnight cultures of pSB1C3-pChr colonies to prepare pSB1C3-pChr for sequence confirmation

Protocols Used: Overnight culture, Patch-plating

Results: N/A

Next Steps: Purification of the recombinant vector for size and sequence confirmation.

Day 5 - 24/07: Purified pSB1C3-pChr for sequence confirmation.

Aim of experiment: Purification of pSB1C3-pChr for sequence confirmation before ligating GFP into it.

Protocols Used:

Plasmid purification, Sequencing

Results: Figure 19 Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.

Next Steps: Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.

Day 6 - 25/07: PCR-amplified ChrB and ChrBopt.

Aim of experiment: Amplification of ChrB and ChrBopt for insertion into pUniprom. Restriction digest of the PCR product with BamHI and PstI for producing compatible sticky ends

Protocols Used: PCR-amplification, Restriction digest

Results: N/A

Next Steps: Ligation and Transformation of ChrB and ChrBopt.

Day 7 - 26/07: Ligated ChrB and ChrBopt into pUniprom.

Aim of experiment: Insertion of ChrB and optimised ChrB into pUniprom vector.

Protocols Used: Ligation

Results: N/A

Next Steps: Transformation of ligated vectors.

Week Beginning 27/07

Summary

Final touches were made on the chromate system. All parts were ligated into their final vectors during this week. Preparations were made for characterisation of each of the parts.

Day 1 - 27/07: Transformed ChrB and ChrBopt into E. coli JM110

Aim of experiment:Transformation of the repressors, ChrB and ChrBopt, into JM110

Protocols Used: Transformation

Results: Figure 20 Plates after transformation.

Next Steps: Purification of pUniprom-ChrB and pUniprom-ChrBopt for sequence confirmation.

Day 2 - 28/07: Digested pSB1C3-pChr for insertion of GFP

Aim of experiment: Digesting sequence confirmed pSB1C3-pChr with SpeI and PstI for subsequent ligation with GFP. Overnight cultures of ChrB and ChrBopt for subsequent plasmid purification, size confirmation, and sequence confirmation.

Protocols Used:

Restriction digest, Ligation of GFP into pSB1C3-pChr.

Overnight culture of of ChrB and ChrBopt.

Next Steps: Transformation of recombinant vector for confirmation of size and sequence.

Results: N/A

Next Steps: Transformation of pSB1C3-pChr-GFP for size and sequence confirmation. Purification of ChrB and ChrBopt for sequence confirmation.

Day 3 - 29/07: Purified ChrB and ChrBopt for size and sequence confirmation.

Aim of experiment: Purification of ChrB and ChrBopt for confirmation of size and sequence.

Protocols Used: Plasmid purification, Restriction digest, Sequencing

Results:

Figure 21 Confirmation of insert size.

Figure 22 Confirmation of sequence.

Next Steps: Confirmation of expression of ChrB and ChrBopt by western blotting.

Day 4 - 30/07: Repeated transformation of pSB1C3-pChr-GFP into E.coli JM110

Aim of experiment: Digest of pSB1C3-pChr with SpeI and PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.

Protocols Used: Restriction digest, Ligation, Transformation

Results: Figure 23 Plates after transformation

Next Steps: Overnight cultures for purification of the pSB1C3-pChr-GFP for confirmation of insert size and sequence.

Day 5 - 31/07: Set up overnight cultures for confirmation of pSB1C3-pChr-GFP.

Aim of experiment: Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.

Day 6 - 01/08: Purified pSB1C3-pChr-GFP for size confirmation and sequencing.

Aim of experiment: Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.

Protocols Used: Plasmid purification, Restriction digest, Sequencing

Results:

Figure 24 Gel for size confirmation.

Figure 25 Sequence confirmation.

Next Steps: Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB and ChrBopt.

Day 7 - 02/08: Set up cultures in preparation for western blotting.

Aim of experiment: Overnight cultures of sequence confirmed ChrB, ChrBopt, pChrGFP1 (from 15/07), pChrGFP3 and pChrGFP4 (31/07) for subsequent western blotting.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Western blotting for characterisation of expression of both, GFP, and ChrB and ChrBopt.

Week Beginning 03/08

Summary

This week was entirely used for characterising the expression of GFP from the promoter pChr, and the expression of ChrB from the tat-promoter in pUniprom.

Day 1 - Day 7 - 03/08 - 09/08: Determined expression of ChrB, ChrBopt, and GFP by western blotting.

Aim of experiment: To determine whether or not the required proteins, GFP, and ChrB, are expressed by blotting against GFP and against a 6His-tag respectively.

Protocols Used: SDS-PAGE and western blot

Results: This procedure was repeated several times during this week in order to find appropriate concentrations and volumes to load. Figure 26 was blotted after SDS-PAGE with the following concentrations:

GFP: Dilute pellet of 1ml overnight culture with 1ml TBS. Take 100µl of this solution and mix with 100µl Laemmli Buffer. Load 3µl on the SDS-gel.
HIS: Dilute pellet of 1ml overnight culture with 200µl Laemmli buffer. Load 10µl on the SDS-gel.

Figure 26

Next Steps:Transformation of both plasmids into MG1655 with the goal to characterise the interaction between ChrB and pChr. Furthermore the original biobrick, BBa_K1058008 will be tested under the same conditions for comparison.

Week Beginning 10/08

Summary

This week was spent with transforming all combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into E. coli MG1655. Tests for their interaction were also prepared.

Day 1 - 10/08: Transformed both pSB1C3-pChr-GFP-plasmids, pUniprom-ChrB, and pUniprom-ChrBopt, as well as BBa_K1058008 into E. coli MG1655.

Aim of experiment: To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into MG1655:

pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (31/07)
pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (15/07)
pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (31/07)
pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (15/07)

in a stepwise fashion. Furthermore transform BBa_K1058008 into MG1655. Transformations were done in a stepwise fashion with only one of the plasmids at a time.

Protocols Used: Plasmid purification, Transformation

Results: Figure 27

Next Steps: Making newly transformed cells competent again in order to transform the second plasmid as well.

Day 2 - 11/08: Set up overnight cultures of all transformed cells.

Aim of experiment: To prepare overnight cultures of yesterdays transformaions in order to make them competent to transform the second plasmid as well.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Make cells of overnight culture competent to transform in second plasmid.

Day 3 - 12/08: Made competent MG1655 with pUniprom-ChrB2, pUniprom-ChrBopt3, pSB1C3-pChr-GFP (15/07), pSB1C3-pChr-GFP (31/07).

Aim of experiment:To make transformed cells competent in order to transform the second plasmid. The same plasmids that were used for the initial transformations were also used for this set of transformations.

Protocols Used: Competent cells, Transformations

Results: N/A

Next Steps: Blot against GFP in order to confirm (or refute) that ChrB interacts with pChr and hence switches off expression of GFP.

Day 4 - 13/08: Transformed pUniprom-ChrB2 and pUniprom-ChrBopt3 into MG1655+pSB1C3-pChr-GFP(15/07) and MG1655+pChrGFP(31/07)

Aim of experiment: To build a strain of E. coli MG1655, containing both plasmids required for a chromate sensor.

Protocols Used: Transformation

Results: No successful transformants were found.

Next Steps: Repeat this experiment but transform pSB1C3-pChr-GFP (15/07) and pSB1C3-pChr-GFP (31/07) into competent MG1655+pUniprom-ChrB2 and MG1655+pUniprom-ChrBopt3 respectively.

Week Beginning 17/08

Summary

During this week E. coli MG1655 containing both plasmids for a. Western Blots were used to determine expression of ChrB, ChrBopt, and GFP.

Day 1 - 17/08: Transformations akin to 13/08 were done, but pSB1C3-pChr-GFP(15/07) and pSB1C3-pChr-GFP(31/07) were transformed into MG1655+pUniprom-ChrB2 and MG1655+pUniprom-ChrBopt3.

Aim of experiment: To build a strain of MG1655, containing both plasmids required for a chromate sensor.

Protocols Used: Transformation

Results: Successful transformants were found on all plates.

Next Steps: To determine expression of ChrB and ChrBopt (via 6His-tag) and expression of GFP.

Day 2 - 18/08: Preparation of samples for western blot.

Aim of experiment: To determine whether ChrB, ChrBopt, and GFP are expressed and have first estimates of differences in expression levels.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Lyse cells and prepare samples for SDS-PAGE + Western-Blot.

Day 3-6 - 19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of ChrB has an effect on the expression of GFP.

Aim of experiment: To determine whether expression of ChrB or ChrBopt has an effect on the expression of GFP.

Protocols Used: SDS-PAGE + Western Blot

Results: Figure 28

Next Steps: Check if different chromate salts have an effect on the respective expression levels.

Week Beginning 24/08

Summary

Plate reader experiments were carried out in order to determine the effect of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄ on the expression of GFP.

Day 1 - Day 7 - 24/08-30/08: Design of Plate reader experiments.

Aim of experiment: To design a series of plate reader experiments to measure OD₆₀₀ and relative fluorescence of the chromate sensor as a response to different concentrations of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄.

Protocols Used: Plate Reader Experiments

Results: Figure 29 MG1655 + BBa_K1058008

Results: Figure 30 MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB

Results: Figure 31 MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt

Results: Figure 32 MG1655 + pSB1C3-pChr-GFP

Next Steps: Try the system with different expression levels of ChrB and ChrBopt. Induce overexpression by exploiting the T7-promoter in pUniprom after transforming the different combinations between pSB1C3-pChr-GFP and pUniprom-ChrB/pUniprom-ChrBopt into BL21(DE3) and adding IPTG.

Week Beginning 31/08

Summary

UK Synbio Conference and UK iGEM meetup at Westminster University.

Week Beginning 07/09

Summary

Parts from CeBiTec Bielefeld arrived and attempts at cloning were made. The chromate sensing system was transformed into BL21(DE3) and some of the plate reader experiments repeated.

Day 1 - 07/09: Transformation of pSB1K3 (Bielefeld) into MG1655.

Aim of experiment: The high-copy plasmid arrived at a concentration of 7 ^ng/_µl. Hence it was transformed in order to produce more of it before purifying and digesting it.

Protocols Used: Transformation,

Overnight culture for new E. coli MG1655

Results: No transformants were found.

Next Steps: Retry transformation with new competent E. coli MG1655.

Day 2 - 08/09: Transformation of pSB1K3 into MG1655.

Aim of experiment: Test transformation of BBa_K1058008 into new MG1655 competent cells and re-transformation of pSB1K3 for subsequent miniprep.

Protocols Used: Transformation

Results: Successful test transformation, but no colonies on pSB1K3 plate.

Next Steps: Use an alternative compatible plasmid for ligation of codon optimised ChrB (Bielefeld).

Day 3 - 09/09: Transformation of pSB1K3 into MG1655.

Aim of experiment: Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested ChrB (Bielefeld) and transformation of pT7KS-ChrB. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations:

pSB1C3-pChr-GFP + pUniprom-ChrB
pSB1C3-pChr-GFP + pUniprom-ChrBopt
pUniprom-ChrB
pUniprom-ChrBopt

Protocols Used: Restriction Digest, Gel Extraction, Ligation

Results:Transformations of the plasmid with pT7KS-ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations of pSB1C3-pChr-GFP + pUniprom-ChrB, pSB1C3-pChr-GFP + pUniprom-ChrBopt, pUniprom-ChrB, and pUniprom-ChrBopt into BL21(DE3) were successful.

Results:

Figure 33 pT7KS-ChrB (Bielefeld) transformations

Figure 34 Transformations into BL21(DE3)

Next Steps: Confirmation of colonies with pT7KS-ChrB by colony PCR. Preparation of recombinant BL21(DE3) for plate reader experiments.

Day 4 - 10/09: Colony PCR of pT7KS-ChrB (Bielefeld).

Aim of experiment: Check if any of the colonies after ligation of ChrB (Bielefeld) into pT7KS were successful despite colonies on the control plate.

Protocols Used: Colony PCR

Results: Figure 34

Next Steps: Prepare transformed BL21(DE3) for plate reader experiments. Attempt new transformation of pT7KS-ChrB (Bielefeld).

Day 5 - 11/09: Preparation for Plate reader experiment with BL21(DE3)

Aim of experiment: Set up overnight cultures for subsequent plate reader experiments.

Protocols Used: Overnight cultures

Results: N/A

Next Steps: Set up plate reader, collect and analyse data. Assess the effect of induction of higher repressor-expression on the level of GFP.

Day 6 - 12/09: Set up plate reader.

Aim of experiment: To design a series of plate reader experiments to measure OD₆₀₀ and relative fluorescence of the chromate sensor. At first no chromate was added to this plate in order to assess the effect of IPTG induction.

Protocols Used: Plate reader experiments

Results: Figure 35

Next Steps:Repeat experiments with added chromate substances.

Week Beginning 14/09

Summary

During this week the samples from the previous plate reader were retrieved and prepared for blotting against both GFP, and the 6His-Tag of ChrB and ChrBopt.

Day 1 - 14/09: Western blot of plate reader samples MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB, and MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt.

Aim of experiment: To identify whether or not there is any detectable difference in both, the level of GFP, and the level of ChrB or ChrBopt, before and after induction with IPTG.

Protocols Used: SDS-PAGE + Western blot

Results: Insert blot image here.

Next Steps: Repeat the assays of the previous weeks by subcloning ChrB, ChrBopt, and ChrB (Bielefeld) into vectors with different expression levels and see how it influences the level of GFP expression. Then add different chromate salts and also probe the system with other substances, e.g. other heavy-metal salts.

Reactants	Volume (µl)
DNA	1
DMSO	2.5
100 µM Forward Primer	0.5
100 µM Reverse Primer	0.5
5 x Herculase Buffer	10
10 mM dNTPs	0.5
Herculase	0.5
H₂O	34.5

	Post Plasmid Purification	Post PCR	gBlock
DNA	1 mg	50 µl	100 ng
Cutsmart Buffer (µl)	6	6	6
Water (µl)	As required	1	As required
Enzyme 1 (µl)	1.5	1.5	1.5
Enzyme 2 (µl)	1.5	1.5	1.5
Total Volume (µl)	30	60	30

	Vector (µl)	2:1 (Vector: Insert) [µl]	3:1 (Vector: Insert) [µl]	4:1 (Vector: Insert) [µl]
T4 DNA Ligase	1	1	1	1
Ligase Buffer	1	1	1	1
Vector	1	1	1	1
Insert	-	2	3	4
H₂0	7	5	4	3

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+      <div class="row">
+        <div class="col-lg-4">
+          <a href="#section1" class="scroll"><span class="glyphicon glyphicon-briefcase"></span></a>
+          <h3>FluID</h3>
+          <p class="about-content">FluID is a cell free, all in one spray which will be used to detect and dinstiguish between body fluids found at crime scenes.</p>
+        </div>
+        <div class="col-lg-4">
+          <a href="#section2"><span class="glyphicon glyphicon-search" type="button"></span></a>
+          <h3>Chromate Biosensor</h3>
+          <p class="about-content">Our chromate biosensor is a device that will be used to detect stainless steel residues left behind on bones in blunt or sharp force trauma cases.</p>
+        </div>
+        <div class="col-lg-4">
+          <a href="#section3"><span class="glyphicon glyphicon-eye-open"></span></a>
+          <h3>Fingerprint Ageing</h3>
+          <p class="about-content">This device will search for the presence of a specifc component found in fingerprint deposits whose presence/absence will indicate approximate fingerprint age.</p>
+        </div>
+      </div>
+    </section>
+<div class="ribbon">
+<div class="rows">
+<!-- WEEK 1 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 22/06/2015</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">This week was all about getting started with the newly arrived biobrick <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008.</a></p>
+         </div>
+         <div id="collapsiblew1" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 22/06: Plated <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> from agar stab.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-1"></span>
+                <div id="collapsiblew1-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To prepare <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> for sequence confirmation, storage, and further processing. The culture was plated from the agar stab and also a liquid culture set up.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Plating, </a>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> and confirmation of size and sequence.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 23/06: Purified <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> from liquid culture.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-2"></span>
+                <div id="collapsiblew1-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To purify <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> for sequence confirmation.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep plasmid purification, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol"> DNA sequencing</a>
+                  </p>
+                  <p><b>Results Sequencing: </b><a data-toggle="modal" data-target="#BBa_K1058008-figure1-modal" class="journal-protocol"> Figure 1 </a></p>
+                  <p><b>Next Steps:</b> PCR of promoter region (<i>pChr</i>) and repressor region (<i>ChrB</i>) of <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> for storage in individual biobricks.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 2 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 29/06</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content"> During this week, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> was disassembled into its constituent parts, <i>pChr</i>, and <i>ChrB</i>.</p>
+         </div>
+         <div id="collapsiblew2" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 29/06: PCR-amplification of <i>pChr</i> and <i>ChrB</i> from <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a>.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew2-1"></span>
+                <div id="collapsiblew2-1" class="collapse box-content">
+                  <p><b>Aim of experiment: </b> To break down <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into the promoter region <i>pChr</i>, and the repressor gene, <i>ChrB</i>, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of <i>ChrB</i> in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of <i>pChr</i> and <i>ChrB</i> into pSB1C3 for submission to registry.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR, </a>
+                    <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction Digest</a>
+                  </p>
+                  <p><b>Results:</b>
+                  <a data-toggle="modal" data-target="#dundee15-chr-igem022-modal" class="journal-protocol"> Figure 2</a>
+                  </p>
+                  <p><b>Next Steps:</b> Repeat of PCR of <i>ChrB</i>. Purification of the Plasmid, containing <a href="http://parts.igem.org/Part:BBa_K1058008">BBa_K1058008</a> and confirmation of size and sequence.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 3 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 06/07</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">During this week ligations of <i>pChr</i> and <i>ChrB</i> into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.</p>
+         </div>
+         <div id="collapsiblew3" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 06/07: Repeated amplification of <i>ChrB</i>. Ligation and transformation of <i>pChr</i> and <i>ChrB</i>.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-1"></span>
+                <div id="collapsiblew3-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of <i>pChr</i> and <i>ChrB</i> into pSB1C3. Transformation of each into a non-pathogenic lab strain of <i>Escherichia coli</i>.
+                  Overnight culture of <i>E. coli DH5-alpha</i>, containing GFPmut2, as a fluorescent reporter for our chromate sensing system</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR-amplification, </a>
+                    <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligation, </a>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures</a>
+                  </p>
+                  <p><b>Results:</b>
+                  <a data-toggle="modal" data-target="#dundee15-chr-igem023-modal" class="journal-protocol"> Figure 3 </a></p>
+                  <p>2 bands were extracted from the gel for further characterisation, the strongest band, hence called <i>ChrBs</i>, and the band directly above, hence called <i>ChrBw</i>. <i>pChr</i>, <i>ChrBs</i>, and <i>ChrBw</i> were ligated into pSB1C3.
+                  pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into <i>E. coli MC1061.</i></p>
+                  <p><b>Next Steps:</b> Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3. Ligation of <i>pChr</i> to <i>GFP coding sequence</i> for construction of a reporter plasmid.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 07/07: Purification of <i>GFP</i> and overnight cultures of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-2"></span>
+                <div id="collapsiblew3-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant <i>E. coli MC1061</i>, containing pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw respectively for further size and sequence confirmation. Overnight culture of pUniprom for subsequent ligation of ChrB</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep, </a>
+                    <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR-amplification, </a>
+                    <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction, </a>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-igem024-modal" class="journal-protocol"> Figure 5 </a> </p>
+                  <p><b>Next Steps:</b> Repeat of amplification of <i>ChrB</i>. Sequence and size confirmation of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3 - 08/07: Purification of plasmids pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw. Size and sequence confirmation.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-3"></span>
+                <div id="collapsiblew3-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of <i>pChr</i> and <i>ChrB</i>. Restriction digestes of all parts in order to produce compatible sticky ends.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep, </a>
+                    <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR amplification, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
+                    <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest</a> of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.</p>
+                  </p>
+                  <p><b>Results:</b><p><a data-toggle="modal" data-target="#dundee15-chr-igem026-modal" class="journal-protocol"> Figure 6 </a> - Size confirmation of <i>pChr</i> and <i>ChrB</i>.</p>
+                  <p><a data-toggle="modal" data-target="#dundee15-chr-pChr1-modal" class="journal-protocol"> Figure 7 </a> - Sequence confirmation of <i>pChr</i></p>
+                  <p><a data-toggle="modal" data-target="#dundee15-chr-ChrBw4-modal" class="journal-protocol"> Figure 8 </a> - Sequence confirmation of <i>ChrB</i>.</p>
+                  </p>
+                  <p><b>Next Steps:</b> Purification and digest of pUniprom for insertion of <i>ChrB</i>.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 4 - 09/07: Digested and cleaned up pUniprom for insertion of <i>ChrB</i>. Amplification of optimised <i>ChrB</i> with primers specific for the insertion into pUniprom.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-4"></span>
+                <div id="collapsiblew3-4" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To prepare pUniprom for insertion of <i>ChrB</i> by digesting it with BamHI and HindIII. To amplify <i>ChrB</i> version with first 15 codons optimised for insertion into pUniprom.</p>
+                  <p><b>Protocols Used:</b>
+                    <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> and
+                    <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol">Gel extraction</a> of pUniprom</p>
+                    <p><a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol">PCR</a> and
+                    <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel extraction </a> of optimised <i>ChrB</i></p>
+                  </p>
+                  <p><b>Results:</b><p><a data-toggle="modal" data-target="#dundee15-chr-igem034-modal" class="journal-protocol"> Figure 9 </a> Gel with pUniprom</p>
+                  <p><a data-toggle="modal" data-target="#dundee15-chr-igem032-modal" class="journal-protocol"> Figure 10 </a>Gel with ChrB</p></p>
+                  <p><b>Next Steps:</b> Ligation of <i>ChrB</i> and codon optimised <i>ChrB</i> into pUniprom.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 5 - 10/07: Ligations of <i>ChrB</i> and optimised <i>ChrB</i> into pUniprom, and of <i>pChr</i> and <i>GFPmut2</i> into pSB1C3.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew3-5"></span>
+                <div id="collapsiblew3-5" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Ligation of <i>ChrB</i> and optimised <i>ChrB</i> into pUniprom via BamHI and HindIII restriction sites. Ligation of <i>pChr</i> and <i>GFPmut2</i> to each other via SpeI/XbaI-link and into pSB1C3 via EcoRI and PstI restriction sites.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligations</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Transformation of abovementioned ligations.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 4 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 13/07</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">During this week successful transformants were identified and propagated.</p>
+         </div>
+         <div id="collapsiblew4" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 13/07: Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-1"></span>
+                <div id="collapsiblew4-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP into <i>E. coli JM110</i> chassis. Setting up a backup restriction digest of pSB1C3 with EcoRI and SpeI in case the double ligation pSB1C3-pChr-GFP is unsuccessful.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformations, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15-chr-first-transformation-modal" class="journal-protocol"> Figure 11 </a></p>
+                  <p><b>Next Steps:</b> Purification of recombinant vectors and confirmation of size and sequence. Store digested pSB1C3 at -20<sup>o</sup>C.</p>
+                </div>
+          </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 14/07: Set up cultures for subsequent purification of recombinant vectors for size and sequence confirmation.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-2"></span>
+                <div id="collapsiblew4-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Purification of recombinant vectors, pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP, for size and sequence confirmation. Patch plating of the same colonies for storage and retrieval once size and sequence have been confirmed.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures, </a>
+                    <a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Patch plating</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Purification of the recombinant plasmids for confirmation of size and sequence.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3 - 15/07: Purified pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight cultures</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-3"></span>
+                <div id="collapsiblew4-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Purification of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight for confirmation of size and sequence. Restriction digest of pUnipromChrB and optimised pUniprom-ChrB optimised with BamHI and HindIII and of pSB1C3-pChr-GFP with EcoRI/PstI for size confirmation. </p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Miniprep, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#colony-modal" class="journal-protocol">Colony PCR, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing </a>
+                  </p>
+                  <p><b>Results:</b>
+                    <p><a data-toggle="modal" data-target="#dundee15-chr-igem036-modal" class="journal-protocol"> Figure 12 </a> Size confirmation of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                    <a data-toggle="modal" data-target="#dundee15-chr-igem037-modal" class="journal-protocol"> Figure 13 </a> Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.</p>
+                    </p><a data-toggle="modal" data-target="#dundee15_chr_pSB1C3-pChr-GFP_reverse" class="journal-protocol"> Figure 14 </a> Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.</p>
+                  </p>
+                  <p><b>Next Steps:</b> Confirmation of sequence of <i>ChrB-opt.</i></p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 4 - 16/07: Set up overnight cultures of colonies chosen after colony-PCR for subsequent purification and sequence confirmation.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-4"></span>
+                <div id="collapsiblew4-4" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Set up overnight cultures of <i>ChrBopt</i> colonies 2, 4, 9, and 12 for sequencing.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures </a></p>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Purification of recombinant plasmids.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 5 - 17/07: Purified and sequenced chosen <i>ChrBopt</i>-colonies.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew4-5"></span>
+                <div id="collapsiblew4-5" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To purify recombinant plasmids for sequence confirmation.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
+                  </p>
+                  <p><b>Results:</b> <i>ChrBopt</i> showed HindIII restriction sites and was hence cleaved into partial inserts.</p>
+                  <p><b>Next Steps:</b> Use alternative cloning protocol for <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                </div>
+          </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 5 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 20/07</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">During this week an alternative cloning strategy for <i>ChrB</i> and <i>ChrBopt</i> was employed. Furthermore, the pSB1C3-pChr-GFP construct was cloned in a stepwise fashion.</p>
+         </div>
+         <div id="collapsiblew5" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 20/07: Went back to the original plate of pSB1C3-pChr-GFP for visualising colonies that do or do not express GFP. </span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-1"></span>
+                <div id="collapsiblew5-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b>To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light. The background of this experiment was that if there is one colony that has both inserts in the correct orientation with respect to each other, then there is perhaps one that also has them with the correct orientation with respect to the plasmid. Colonies identified as expressing GFP were patch plated and grown in liquid cultures over night in order to confirm the sequence of the insert.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#plating-modal" class="journal-protocol"> Patch-plating, </a>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> overnight cultures</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_pChrGFPplates_revisited" class="journal-protocol"> Figure 15</a> pSB1C3-pChr-GFP in <i>E. coli JM110</i>.</p>
+                  <p><b>Next Steps:</b> Sequence confirmation of newly selected colonies.</p>
+                </div>
+          </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 21/07: Purified plasmids from new colonies and confirmed sequence. Restriction digest of pUniprom with BamHI and PstI.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-2"></span>
+                <div id="collapsiblew5-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b>Purification of newly selected colonies, 1, 5, 6, and 7, containing pSB1C3-pChr-GFP for subsequent sequence confirmation. Restriction digest of pUniprom with BamHI and PstI to employ an alternative cloning strategy of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                  <p><b>Protocols Used:</b>
+                    <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Miniprep </a> and
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing </a> of pChrGFP insert.</p>
+                    <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest</a> of pUniprom.
+                  </p>
+                  <p><b>Results:</b>
+                  <p><a data-toggle="modal" data-target="#dundee15_chr_pChrGFP_revisited_seq" class="journal-protocol"> Figure 16 </a> Alignment of sequencing results.</p>
+                  <p><a data-toggle="modal" data-target="#dundee15_chr_igem039" class="journal-protocol"> Figure 17 </a> Gel after restriction digest of pUniprom.</p>
+                  <p><b>Next Steps:</b>Stepwise ligation of <i>pChr</i> and <i>GFP</i> into pSB1C3 in order to clone the inserts in the correct orientation with respect to the plasmid. Insertion of <i>ChrB</i> and <i>ChrBopt</i> into pUniprom via BamHI and PstI links.</p>
+                </div>
+          </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3 - 22/07: Ligated <i>pChr</i> into pSB1C3 via EcoRI and SpeI sites and cloned the recombinant plasmid into <i>E. coli JM110</i>.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-3"></span>
+                <div id="collapsiblew5-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Ligation of <i>pChr</i> into pSB1C3 via EcoRI SpeI link. This is the first step of a 2-step cloning strategy with the aim of cloning <i>pChr</i> and <i>GFP</i> into pSB1C3 in the correct orientation with respect to the plasmid.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligation, </a>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150723_pSB1C3-pChr" class="journal-protocol"> Figure 18 </a> Plates after transformation of pChr into pSB1C3.</p>
+                  <p><b>Next Steps:</b> Sequence confirmation of pChr in pSB1C3.</p>
+                </div>
+         </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 4 - 23/07: Set up overnight cultures of pSB1C3-pChr colonies.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew1-4"></span>
+                <div id="collapsiblew1-4" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Set up overnight cultures of pSB1C3-pChr colonies to prepare pSB1C3-pChr for sequence confirmation</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture, </a>
+                    <a data-toggle="modal" data-target="#plating-modal" class="journal-protocol">Patch-plating</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Purification of the recombinant vector for size and sequence confirmation.</p>
+                </div>
+          </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 5 - 24/07: Purified pSB1C3-pChr for sequence confirmation.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-5"></span>
+                <div id="collapsiblew5-5" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr for sequence confirmation before ligating <i>GFP</i> into it.</p>
+                  <p><b>Protocols Used:</b>
+                    <p><a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol"> Plasmid purification, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing </a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150727_pSB1C3-pChr_alignment" class="journal-protocol"> Figure 19 </a> Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.</p>
+                  <p><b>Next Steps:</b> Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 6 - 25/07: PCR-amplified <i>ChrB</i> and <i>ChrBopt</i>.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-6"></span>
+                <div id="collapsiblew5-6" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Amplification of <i>ChrB</i> and <i>ChrBopt</i> for insertion into pUniprom. Restriction digest of the PCR product with BamHI and PstI for producing compatible sticky ends</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#PCR-modal" class="journal-protocol"> PCR-amplification, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction digest</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Ligation and Transformation of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                </div>
+          </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 7 - 26/07: Ligated <i>ChrB</i> and <i>ChrBopt</i> into pUniprom.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew5-7"></span>
+                <div id="collapsiblew5-7" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Insertion of ChrB and optimised ChrB into pUniprom vector.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligation</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Transformation of ligated vectors.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 6 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 27/07</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">Final touches were made on the chromate system. All parts were ligated into their final vectors during this week. Preparations were made for characterisation of each of the parts.</p>
+         </div>
+         <div id="collapsiblew6" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 27/07: Transformed <i>ChrB</i> and <i>ChrBopt</i> into <i>E. coli JM110</i></span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-1"></span>
+                <div id="collapsiblew6-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b>Transformation of the repressors, <i>ChrB</i> and <i>ChrBopt</i>, into <i>JM110</i></p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150728_pUniprom-ChrB" class="journal-protocol"> Figure 20</a> Plates after transformation.</p>
+                  <p><b>Next Steps:</b> Purification of pUniprom-ChrB and pUniprom-ChrBopt for sequence confirmation.</p>
+                </div>
+         </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 28/07: Digested pSB1C3-pChr for insertion of <i>GFP</i></span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-2"></span>
+                <div id="collapsiblew6-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Digesting sequence confirmed pSB1C3-pChr with SpeI and PstI for subsequent ligation with <i>GFP</i>. Overnight cultures of <i>ChrB</i> and <i>ChrBopt</i> for subsequent plasmid purification, size confirmation, and sequence confirmation.</p>
+                  <p><b>Protocols Used:</b>
+                    <p><a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation</a> of <i>GFP</i> into pSB1C3-pChr.</p>
+                    <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a> of of <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                  </p>
+                  <p><b>Next Steps:</b> Transformation of recombinant vector for confirmation of size and sequence.</p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Transformation of pSB1C3-pChr-GFP for size and sequence confirmation. Purification of <i>ChrB</i> and <i>ChrBopt</i> for sequence confirmation.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3 - 29/07: Purified <i>ChrB</i> and <i>ChrBopt</i> for size and sequence confirmation.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-3"></span>
+                <div id="collapsiblew6-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Purification of <i>ChrB</i> and <i>ChrBopt</i> for confirmation of size and sequence.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid purification, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
+                  </p>
+                  <p><b>Results:</b>
+                    <p><a data-toggle="modal" data-target="#dundee15_chr_igem040" class="journal-protocol">Figure 21 </a>Confirmation of insert size.</p>
+                    <p><a data-toggle="modal" data-target="#dundee15_chr_150729_chrb-combined" class="journal-protocol"> Figure 22 </a>Confirmation of sequence.</p>
+                  </p>
+                  <p><b>Next Steps:</b> Confirmation of expression of <i>ChrB</i> and <i>ChrBopt</i> by western blotting.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 4 - 30/07: Repeated transformation of pSB1C3-pChr-GFP into <i>E.coli JM110</i></span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-4"></span>
+                <div id="collapsiblew6-4" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Digest of pSB1C3-pChr with SpeI and PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.</p>
+                  <p><b>Protocols Used: </b>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol">Ligation, </a>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150731_pChrGFP-ligations" class="journal-protocol"> Figure 23 </a>Plates after transformation</p>
+                  <p><b>Next Steps:</b> Overnight cultures for purification of the pSB1C3-pChr-GFP for confirmation of insert size and sequence.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 5 - 31/07: Set up overnight cultures for confirmation of pSB1C3-pChr-GFP.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-5"></span>
+                <div id="collapsiblew6-5" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Purification of the Plasmids for confirmation of insert size and sequence.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 6 - 01/08: Purified pSB1C3-pChr-GFP for size confirmation and sequencing.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-6"></span>
+                <div id="collapsiblew6-6" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid purification, </a>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol">Restriction digest, </a>
+                    <a data-toggle="modal" data-target="#sequencing-modal" class="journal-protocol">Sequencing</a>
+                  </p>
+                  <p><b>Results:</b>
+                    <p><a data-toggle="modal" data-target="#dundee15_chr_igem043" class="journal-protocol"> Figure 24 </a> Gel for size confirmation.</p>
+                    <p><a data-toggle="modal" data-target="#dundee15_chr_150801_pchrgfp" class="journal-protocol"> Figure 25 </a> Sequence confirmation.</p>
+                  </p>
+                  <p><b>Next Steps:</b> Overnight cultures in preparation for western blotting for characterisation of expression of both, <i>GFP</i>, and <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 7 - 02/08: Set up cultures in preparation for western blotting.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew6-7"></span>
+                <div id="collapsiblew6-7" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Overnight cultures of sequence confirmed <i>ChrB</i>, <i>ChrBopt</i>, <i>pChrGFP1</i> (from 15/07), <i>pChrGFP3</i> and <i>pChrGFP4</i> (31/07) for subsequent western blotting.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Western blotting for characterisation of expression of both, <i>GFP</i>, and <i>ChrB</i> and <i>ChrBopt</i>.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 7 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 03/08</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew7"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">This week was entirely used for characterising the expression of GFP from the promoter <i>pChr</i>, and the expression of <i>ChrB</i> from the <a href="http://parts.igem.org/Part:BBa_K895000" target="_blank"><i>tat-promoter</i></a> in pUniprom.</p>
+         </div>
+         <div id="collapsiblew7" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - Day 7 - 03/08 - 09/08: Determined expression of <i>ChrB</i>, <i>ChrBopt</i>, and GFP by western blotting.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew7-1"></span>
+                <div id="collapsiblew7-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To determine whether or not the required proteins, <i>GFP</i>, and <i>ChrB</i>, are expressed by blotting against GFP and against a 6His-tag respectively.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol">SDS-PAGE and western blot</a>
+                  </p>
+                  <p><b>Results:</b> This procedure was repeated several times during this week in order to find appropriate concentrations and volumes to load. Figure 26 was blotted after SDS-PAGE with the following concentrations:
+                    <ul>
+                      <li>GFP: Dilute pellet of 1ml overnight culture with 1ml TBS. Take 100µl of this solution and mix with 100µl Laemmli Buffer. Load 3µl on the SDS-gel.</li>
+                      <li>HIS: Dilute pellet of 1ml overnight culture with 200µl Laemmli buffer. Load 10µl on the SDS-gel.</li>
+                    </ul>
+                  </p>
+                  <p><a data-toggle="modal" data-target="#dundee15_chr_150809_blot" class="journal-protocol"> Figure 26 </a></p>
+                  <p><b>Next Steps:</b>Transformation of both plasmids into MG1655 with the goal to characterise the interaction between <i>ChrB</i> and <i>pChr</i>. Furthermore the original biobrick, <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> will be tested under the same conditions for comparison.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 8 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 10/08</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew8"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">This week was spent with transforming all combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into <i>E. coli MG1655</i>. Tests for their interaction were also prepared.</p>
+         </div>
+         <div id="collapsiblew8" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 10/08: Transformed both pSB1C3-pChr-GFP-plasmids, pUniprom-ChrB, and pUniprom-ChrBopt, as well as <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into <i>E. coli MG1655</i>.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew8-1"></span>
+                <div id="collapsiblew8-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into <i>MG1655</i>:
+                    <ul>
+                      <li>pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (31/07)</li>
+                      <li>pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (15/07)</li>
+                      <li>pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (31/07)</li>
+                      <li>pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (15/07)</li>
+                    </ul>
+                  in a stepwise fashion. Furthermore transform <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a> into <i>MG1655</i>. Transformations were done in a stepwise fashion with only one of the plasmids at a time.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#miniprep-modal" class="journal-protocol">Plasmid purification, </a>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformation</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150811_transformations" class="journal-protocol"> Figure 27 </a></p>
+                  <p><b>Next Steps:</b> Making newly transformed cells competent again in order to transform the second plasmid as well.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 11/08: Set up overnight cultures of all transformed cells.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew8-2"></span>
+                <div id="collapsiblew8-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To prepare overnight cultures of yesterdays transformaions in order to make them competent to transform the second plasmid as well.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol">Overnight culture</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Make cells of overnight culture competent to transform in second plasmid.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3 - 12/08: Made competent <i>MG1655</i> with pUniprom-ChrB2, pUniprom-ChrBopt3, pSB1C3-pChr-GFP (15/07), pSB1C3-pChr-GFP (31/07).</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew8-3"></span>
+                <div id="collapsiblew8-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b>To make transformed cells competent in order to transform the second plasmid. The same plasmids that were used for the initial transformations were also used for this set of transformations.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#competent-modal" class="journal-protocol">Competent cells, </a>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol">Transformations </a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Blot against GFP in order to confirm (or refute) that <i>ChrB</i> interacts with <i>pChr</i> and hence switches off expression of GFP.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 4 - 13/08: Transformed pUniprom-ChrB2 and pUniprom-ChrBopt3 into <i>MG1655+pSB1C3-pChr-GFP</i>(15/07) and <i>MG1655+pChrGFP</i>(31/07)</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew8-4"></span>
+                <div id="collapsiblew8-4" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To build a strain of <i>E. coli MG1655</i>, containing both plasmids required for a chromate sensor.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation</a>
+                  </p>
+                  <p><b>Results:</b> No successful transformants were found.</p>
+                  <p><b>Next Steps:</b> Repeat this experiment but transform pSB1C3-pChr-GFP (15/07) and pSB1C3-pChr-GFP (31/07) into competent <i>MG1655+pUniprom-ChrB2</i> and <i>MG1655+pUniprom-ChrBopt3</i> respectively.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 9 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 17/08</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">During this week <i>E. coli MG1655</i> containing both plasmids for a. Western Blots were used to determine expression of <i>ChrB</i>, <i>ChrBopt</i>, and GFP.</a></p>
+         </div>
+         <div id="collapsiblew9" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 17/08: Transformations akin to 13/08 were done, but pSB1C3-pChr-GFP(15/07) and pSB1C3-pChr-GFP(31/07) were transformed into <i>MG1655+pUniprom-ChrB2</i> and <i>MG1655+pUniprom-ChrBopt3</i>.</span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9-1"></span>
+                <div id="collapsiblew9-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To build a strain of MG1655, containing both plasmids required for a chromate sensor.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation </a>
+                  </p>
+                  <p><b>Results:</b> Successful transformants were found on all plates.</p>
+                  <p><b>Next Steps:</b> To determine expression of <i>ChrB</i> and <i>ChrBopt</i> (via 6His-tag) and expression of GFP.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 18/08: Preparation of samples for western blot.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9-2"></span>
+                <div id="collapsiblew9-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To determine whether <i>ChrB</i>, <i>ChrBopt</i>, and GFP are expressed and have first estimates of differences in expression levels.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Lyse cells and prepare samples for SDS-PAGE + Western-Blot.</p>
+                </div>
+        </div>
+       </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3-6 - 19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of <i>ChrB</i> has an effect on the expression of GFP.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew9-3"></span>
+                <div id="collapsiblew9-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To determine whether expression of <i>ChrB</i> or <i>ChrBopt</i> has an effect on the expression of GFP.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> SDS-PAGE + Western Blot</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150822_blot" class="journal-protocol"> Figure 28 </a></p>
+                  <p><b>Next Steps:</b> Check if different chromate salts have an effect on the respective expression levels.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 10 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 24/08</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew10"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">Plate reader experiments were carried out in order to determine the effect of K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub> on the expression of GFP.</a></p>
+         </div>
+         <div id="collapsiblew10" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - Day 7 - 24/08-30/08: Design of Plate reader experiments.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew10-1"></span>
+                <div id="collapsiblew10-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To design a series of plate reader experiments to measure OD<sub>600</sub> and relative fluorescence of the chromate sensor as a response to different concentrations of K<sub>2</sub>CrO<sub>4</sub>, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>, CrCl<sub>3</sub>, and K<sub>2</sub>SO<sub>4</sub>.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#platereader-modal" class="journal-protocol"> Plate Reader Experiments </a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_BBa_K1058008" class="journal-protocol"> Figure 29 </a><i>MG1655 + <a href="http://parts.igem.org/Part:BBa_K1058008" target="_blank">BBa_K1058008</a></i></p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#icecream" class="journal-protocol"> Figure 30 </a><i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB</i></p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#flake" class="journal-protocol"> Figure 31 </a><i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt</i></p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150824_platereader_pchrbgfp" class="journal-protocol"> Figure 32 </a><i>MG1655 + pSB1C3-pChr-GFP</i></p>
+                  <p><b>Next Steps:</b> Try the system with different expression levels of <i>ChrB</i> and <i>ChrBopt</i>. Induce overexpression by exploiting the T7-promoter in pUniprom after transforming the different combinations between pSB1C3-pChr-GFP and pUniprom-ChrB/pUniprom-ChrBopt into <i>BL21(DE3)</i> and adding IPTG.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 11 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 31/08</div>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">UK Synbio Conference and UK iGEM meetup at Westminster University.</a></p>
+         </div>
+         <div id="collapsiblew11" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+         </div>
+     </div>
+    </div>
+  </div>
+<!-- WEEK 12 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 07/09</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">Parts from CeBiTec Bielefeld arrived and attempts at cloning were made. The chromate sensing system was transformed into <i>BL21(DE3)</i> and some of the plate reader experiments repeated.</a></p>
+         </div>
+         <div id="collapsiblew12" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 07/09: Transformation of pSB1K3 (Bielefeld) into <i>MG1655</i>.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-1"></span>
+                <div id="collapsiblew12-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> The high-copy plasmid arrived at a concentration of 7 <sup>ng</sup>/<sub>µl</sub>. Hence it was transformed in order to produce more of it before purifying and digesting it.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation, </a>
+                    <p><a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight culture</a> for new <i>E. coli MG1655</i></p>
+                  </p>
+                  <p><b>Results:</b> No transformants were found.</p>
+                  <p><b>Next Steps:</b> Retry transformation with new competent <i>E. coli MG1655</i>.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 2 - 08/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-2"></span>
+                <div id="collapsiblew12-2" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Test transformation of BBa_K1058008 into new <i>MG1655</i> competent cells and re-transformation of pSB1K3 for subsequent miniprep.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#transformation-modal" class="journal-protocol"> Transformation </a>
+                  </p>
+                  <p><b>Results:</b> Successful test transformation, but no colonies on pSB1K3 plate.</p>
+                  <p><b>Next Steps:</b> Use an alternative compatible plasmid for ligation of codon optimised <i>ChrB</i> (Bielefeld).</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 3 - 09/09: Transformation of pSB1K3 into MG1655.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-3"></span>
+                <div id="collapsiblew12-3" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested <i>ChrB</i> (Bielefeld) and transformation of pT7KS-ChrB. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations: <ul>
+                                  <li>pSB1C3-pChr-GFP + pUniprom-ChrB</li>
+                                  <li>pSB1C3-pChr-GFP + pUniprom-ChrBopt</li>
+                                  <li>pUniprom-ChrB</li>
+                                  <li>pUniprom-ChrBopt</li>
+                                </ul>
+                  </p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#restriction-digest-modal" class="journal-protocol"> Restriction Digest, </a>
+                    <a data-toggle="modal" data-target="#gelextraction-modal" class="journal-protocol"> Gel Extraction, </a>
+                    <a data-toggle="modal" data-target="#ligation-modal" class="journal-protocol"> Ligation </a>
+                  </p>
+                  <p><b>Results:</b>Transformations of the plasmid with pT7KS-ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations of pSB1C3-pChr-GFP + pUniprom-ChrB, pSB1C3-pChr-GFP + pUniprom-ChrBopt, pUniprom-ChrB, and pUniprom-ChrBopt into <i>BL21(DE3)</i> were successful.</p>
+                  <p><b>Results:</b>
+                    <p><a data-toggle="modal" data-target="#dundee15_chr_150911_chrbbielefeld" class="journal-protocol"> Figure 33 </a> pT7KS-ChrB (Bielefeld) transformations</p>
+                    <p><a data-toggle="modal" data-target="#dundee15_chr_150911_bl21" class="journal-protocol"> Figure 34 </a> Transformations into <i>BL21(DE3)</i></p>
+                  </p>
+                  <p><b>Next Steps:</b> Confirmation of colonies with pT7KS-ChrB by colony PCR. Preparation of recombinant <i>BL21(DE3)</i> for plate reader experiments.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 4 - 10/09: Colony PCR of pT7KS-ChrB (Bielefeld).</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-4"></span>
+                <div id="collapsiblew12-4" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Check if any of the colonies after ligation of ChrB (Bielefeld) into pT7KS were successful despite colonies on the control plate.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#colony-modal" class="journal-protocol"> Colony PCR </a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_150911_igem050" class="journal-protocol"> Figure 34 </a></p>
+                  <p><b>Next Steps:</b> Prepare transformed <i>BL21(DE3)</i> for plate reader experiments. Attempt new transformation of pT7KS-ChrB (Bielefeld).</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 5 - 11/09: Preparation for Plate reader experiment with <i>BL21(DE3)</i></span>
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-5"></span>
+                <div id="collapsiblew12-5" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> Set up overnight cultures for subsequent plate reader experiments.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#overnightculture-modal" class="journal-protocol"> Overnight cultures </a>
+                  </p>
+                  <p><b>Results:</b> N/A</p>
+                  <p><b>Next Steps:</b> Set up plate reader, collect and analyse data. Assess the effect of induction of higher repressor-expression on the level of GFP.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 6 - 12/09: Set up plate reader.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew12-6"></span>
+                <div id="collapsiblew12-6" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To design a series of plate reader experiments to measure OD<sub>600</sub> and relative fluorescence of the chromate sensor. At first no chromate was added to this plate in order to assess the effect of IPTG induction.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#platereader-modal" class="journal-protocol"> Plate reader experiments</a>
+                  </p>
+                  <p><b>Results:</b><a data-toggle="modal" data-target="#dundee15_chr_platereaderinduced" class="journal-protocol"> Figure 35 </a></p>
+                  <p><b>Next Steps:</b>Repeat experiments with added chromate substances.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>
+<!-- WEEK 13 -->
+  <div class="row">
+    <div class="border-week">
+     <div class="box">
+        <div class="labtitle">Week Beginning 14/09</div>
+         <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew13"></span>
+          <div class="box-content">
+          <p class="journal-summary-heading">Summary</p>
+         <p class="journal-content">During this week the samples from the previous plate reader were retrieved and prepared for blotting against both GFP, and the 6His-Tag of <i>ChrB</i> and <i>ChrBopt</i>.</a></p>
+         </div>
+         <div id="collapsiblew13" class="collapse week-content">
+         <!-- when copying for next week, make sure to change labtitle, span, collapsible in last line. In each separate day change span and following div-->
+      <div class="row">
+       <div class="border-day">
+        <div class="box">
+            <span class="box-title">Day 1 - 14/09: Western blot of plate reader samples <i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB</i>, and  <i>MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt</i>.</span> <!--Title of collapsible box-->
+              <span type="button" class="chevron_toggleable box-btn glyphicon glyphicon-chevron-down" data-toggle="collapse" data-target="#collapsiblew13-1"></span>
+                <div id="collapsiblew13-1" class="collapse box-content">
+                  <p><b>Aim of experiment:</b> To identify whether or not there is any detectable difference in both, the level of GFP, and the level of ChrB or ChrBopt, before and after induction with IPTG.</p>
+                  <p><b>Protocols Used:</b>
+                    <a data-toggle="modal" data-target="#sds-modal" class="journal-protocol"> SDS-PAGE + Western blot </a>
+                  </p>
+                  <p><b>Results:</b> Insert blot image here.</p>
+                  <p><b>Next Steps:</b> Repeat the assays of the previous weeks by subcloning ChrB, ChrBopt, and ChrB (Bielefeld) into vectors with different expression levels and see how it influences the level of GFP expression. Then add different chromate salts and also probe the system with other substances, e.g. other heavy-metal salts.</p>
+                </div>
+        </div>
+        </div>
+      </div>
+         </div>
+     </div>
+   </div>
+  </div>