Team:Uppsala-Sweden/Notebook

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Notebook

Week 1

This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See the protocols SOB-medium, LB medium and competent cell preparation). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C. We received the pTJ122 plasmid carrying the ccaS, ccaR and cph8 genes as well as the PcpcG2 promoter from Christopher A Voigt at University of California San Francisco.

Week 2

2011-06-27

This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for transforming TOP10 competent cells. The result was good, we measured a competence efficiency of 1.7 * 10^8 transformants /ug DNA.

Blue / green output

The strains carrying the amilGFP (green/yellow output) and amilCP (blue output) plasmids were provided by J.F Miller, UCLA. They arrived on plates witch were malhandled during the delivery. Both colors looked really nice but since the colonies were all mixed into each other we started by re-plating to obtain single colonies.

2011-06-28

Blue / green output

Started overnight cultures of E coli carrying the plasmids pGEM11-amilGFP and pGEM14-amilCP. We followed the protocol for overnight culture and glycerol stock for the initial preparations on the output modules.

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):
pSB1A3-J04450 (ampR backbone) – Lei & Sibel
pSB1C3-J04450 (CmR backbone) – Lei & Sibel
pSB1K3-J04450 (KanR backbone) – Lie & Sibel
pSB1AK3-B0014 (Double terminator) –Laura & Pikkei
pSB1AK3-B1001 (synthetic terminator) – Karl &Hamid

pSB1A2-B0034 (Standard RBS) - Karl &Hamid
pSB2K3-I15008 (ho1, chromophore synthesis gene)- Mohammed, Erik L & Tomas
pSB2K3-I15009 (pcyA, chomophore synthesis gene) - Mohammed, Erik L &Tomas
pSB1A2-R0011 (PLlacO, lacI repressable promotor) -–Laura & Pekkie
pSB2K3-I15010 (cph8 red sensor) – Lei & Sibel

2011-06-29

Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):
pSB2K3-Q03530 (cII inverter) - Tomas, Erik L, Pikkei, Lidaw
pSB1AK3-B0015 (Double terminator) - Antonio, Lei, Sibel, Cherno
pSB2K3-Q01511 (cI inverter) – Laura & Hamid
pSB1A2-R0082 (PompC) - Tomas, Erik L, Pikkei, Lidaw
pSB1A2-K093005 (RBS + RFP, red pigment) - Laura & Hamid

Streaked on plate
pSB3T5-J04450 (low copy vector tet, ori P15A) – From Erik G

Evaluation: All samples were good. pSB1K3 plates with RFP inserts are less red than pSB1A3 and pSB1C3. Re-streaking of the successful transformations from 2011-07-28. The same people who did the transformation proceeded with re-streaking. Cph8 had no colonies at all, neither on the 1x or 10X dilution plate. Apparently there seems to be a problem with cph8. Thus, cph8 (red sensor) had to be redone due to failure in the first attempt.

2011-06-30

Re-streaking of the transformants from 11-06-29 and started overnight cultures from the re-streaked plates from the previous day (11-06-28).

Evaluation: Most transformations succeeded. However, cI inverter gave very little amount of colonies and cph8 failed again. It’s time to use another approach. We got one single colony on the transformation plates and we will screen that one, but if that colony is wrong will try to run a PCR using the standard VF2 and VR primers using the DNA in the kit as template. If there is any BioBrick plasmid in that well, we should get a PCR product. If this will not work out, we need to either synthesize the gene or to PCR up the gene from the Voigt plasmid pJT122. We will then need to clone it into a BioBrick plasmid and run site-directed mutagenesis on it to eliminate the illegal PstI restriction site. Since cph8 is an important part of our project we have to solve this dilemma.

2011-07-01

Started by doing overnight cultures from the re-streaked plates from 11-06-30. After that Lei and Sibel sterile filtered 20 % glycerol for making glycerol stocks. All the overnight cultures prepared 11-06-30 were frozen in -80°C.

2011-07-02

All the overnight cultures prepared 11-07-01 were mixed with glycerol and frozen in -80°C. At this stage content with the work done this far.

Week 3

2011-07-04

Started by doing overnight cultures (3 ml in selective LB) of the strains carrying these plasmids: pSB1A3-J04450 (vector, ampR)
pSB1C3-J04450 (vector, CmR)
pSB1K3-J04450 (vector, KanR)
pSB1A2-B0034 (Standard RBS)
pSB2K3-I15008 (ho1, chromophore synthesis gene)
pSB2K3-I15009 (pcyA, chomophore synthesis gene)
pSB2K3-Q03530 (cII inverter)
pSB1AK3-B0015 (Double terminator)
pSB2K3-Q01511 (cI inverter)
pSB1A2-R0082 (PompC)

The purpose was to make plasmid preparation the day after.

2011-07-05

Green sensor
The day started off by running BioBrick overhang PCR on the DNA template of the green light sensor, which includes the two genes ccaR, ccaS and the PcpcG2 promotor (see protocol ccaR BioBrick, protocol ccaS BioBrick & protocol PcpcG2 BioBrick). Voigt(UCSF) provided the green sensor, the red sensor and PcpcG2 on plasmid pJT122 (11,088 bp) http://www.ncbi.nlm.nih.gov/pubmed/21035461 Tabor et al 2011.

Green & Blue output
Site-specific mutagenesis was performed on amilGFP and amilCP in order to remove illegal EcoRI sites within the genes (protocol amilGFP_EcoRI, protocol amilCP_EcoRI).

After lunch we did a plasmid preparation of the overnight cultures from 2011-07-04.

Red sensor
At the end of the day we performed colony PCR to verify the length of the single clone we got of red light sensor (cph8) strain.

2011-07-06

Preparation of agarose gel (1%) for the gel electrophoresis. Then we ran our PCR products from 11-07-05 on the gel.

Finally we initiated the first BioBrick assembly. The following entities were assembled:

Chromophore
RBS (B0034) + pcyA (I15009) - Lidaw, Pikkei & Johanna
RBS (B0034) +ho1 (I15008 ) - Erik L & Tomas

Red output
PompC (R0082) + Inv.cl (Q01511) – Kalle, Hamid, Ismael

The plasmids were cut, the enzymes heat inactivated. After mixing the upstream parts, the downstream parts and the backbones, T4 ligase was added and the ligation mixes were left over night in room temperature.

Evalutation: We couldn’t observe any band on the gel corresponding to cph8. Thus the transformation of cph8 has failed completely. We came up with the following conclusions: either there were no or little DNA in the iGEM kit, or the kit just contained something else.

All the other gels were successful but the band for pcpcG2 was slightly weak. The gel bands for ccaR and ccaS looked good. Hence, we have successfully PCR amplified the green sensor parts. Also, the PCR products from the site directed mutagenesis of amilCP_EcoRI and amilGFP_EcoRI showed strong and clear bands at the expected size. Furthermore we can now proceed on cloning ccaR, ccaS and the PcpcG2.

2011-07-07

Re-circularized of pigment vectors after site directed mutagenesis by Sibel. The PCR product was purified, phosphorylated with PNK, template plasmid was degraded using DpnI and finally the linear plasmid was re-circularized using T4 ligase (PCR purification protocol, Phosphorylation of DNA, DpnI digestion protocol).

Tomas and Antonio performed transformation of assembly RBS-ho1 (Protocol for transforming TOP10 competent cells). Transformation of mutagenized amilCP_EcoRI and amilGFP_EcoRI by Hamid and Lei.

Digestion of ccaS and ccaR PCR products and plasmid backbone plasmid pSB1C3 by Tomas and Antonio followed up by cloning and transformation by Erik L (BioBrick Assembly Manual). Lidaw And Johanna transformed the RBS-PcyA assembly.

2011-07-08

The transformation from yesterday was successful; hence the same people could proceed with restreaking the transformants. Hamid and Tomas re-did PCR on pcpcG2 using primers with BioBrick overhangs because of the weak band we got last time. This time we got a stronger band.

λ Red

After internal discussions concerning progresses this far, we decided to start preparing the λ Red assembly parallel with the other assemblies. We need to knock out the gene envZ in our E coli strain to avoid cross-talk with our red sensor cph8. To do this, we will use Lambda Red recombineering http://www.ncbi.nlm.nih.gov/pubmed/10829079 Datsenko and Wanner, 2000.

First step was to perform colony PCR of a strain carrying the plasmid pKD4 (Datsenko) to generate a PCR fragment with a kanamycin resistance cassette flanked by FRT sites and homologies to envZ: “EnvZ- Knock out FRT-Kan” (PCR protocol of envZ knockout FRT-Kan_FRT). We could observe the bands around 1477 bp as we expected. However, we also got a stronger band for the negative control (NC). Even though it seems like we got the right product, the appearance of a band in the NC force us to redo the experiment. Just as a precautious measure.

2011-07-10

Today we screened the transformants from 11-07-08
3A assemblies:
RBS-pcyA,
RBS-ho1
PompC-Inv.cl,
Clonings:
ccaS and ccaR.
Mutagenesis:
amilCP and amilGFP.
There were 6 clones of each transformants, 42 samples in total.

We selected and suspended one colony from each re-streaked clone in 20-30 µl of PBS. We ran colony PCR from of each suspended colony using taq polymerase using the VF2 and VR primers. We also started overnight cultures in selective medium from the suspensions, 1 ml in each.

As a final attempt to get the cph8 gene from the iGEM kit, we tried running a PCR using the VF2 and VR primers and DNA from the kit as template. To be able to get high quality on the PCR product, we used Phusion DNA polymerase.

Last thing was to run an agarose gel of the PCR products. We also ran the PCR product of the envZ Knockout cassette and cph8 on agarose gel. The envZ Knockout had a clear band (we lowered the annealing temperature from 60°C to 59°C). Last thing we did was doing PCR purification of envZ Knockout (Purification protocol). The cph8 showed no bands on the gel, indicating that there simply is no BioBrick plasmid DNA in the kit well.

Week 4

2011-07-11

Today we did a frozen stock of all overnight cultures in -80°C freezer (ccaS clone 1,RBS-pcyA, amilGFP, amilCP, ccaR, RBS-ho1, PompC-Inv.cI). We also started 3 ml cultures by inoculating them with 100 µl of the overnight cultures of the two clones of ccaR, RBS-pcyA and RBS-ho1, the constructs needed for the upcoming assemblies. When the cultures had grown for about 4 hours, we did plasmid preparations of them. We did PCR of amilCP using the pGEM14-amilCP_EcoRI plasmid as template and using primers to add BioBrick prefix and suffix. Mutagensis of amilGFP to remove PstI site and ccaS_EcoRI mutagenesis, round one.

After studying the experience section on the Registry’s homepage, we decided to change inverters to parts Q04400 (TetR inverter) and Q04510 (cI inverter). The inverters we originally chose were not so well characterized, and the promoters they used as output might be too weak for our system.

Transformation of parts Q04400 (TetR inverter) and Q04510 (cI inverter).

2011-07-12

The day started by running a gel of the mutagenized ccaS, amilCP-BB10 PCR product, and amilGFP_PstI linear plasmid. At the same time we prepared the samples (ccaR, ccaS, RBS-ho1 and RBS-pcyA) for sequencing. Plating of strains carrying plasmids with terminator B1001, PLlacO, pSB3T5 and pSB4K5. Purification of PCR products of ccaS_EcoRI, amilGFP_PstI, amilCP_BB10, and PcpcG2. Re-ligation of mutagenized ccaS and amilGFP, and cloning of amilCP_BB10.

The transformation of inverters from yesterday didn’t go well. The lambda-cI-inverter had only a few colonies, while the tetR inverter had no colonies at all. So TetR inverter has to be redone. This time the transformation used 2 µl of DNA from the MIT distribution. Re-streak of cI inverter transformants from 2011-07-11.

2011-07-13

We started the day by running EnvZ-KO, ccaS, EcoRI- digested amilCP and amilGFP on a gel. We obtained good results; there were no distinguishable multiple bands on the gel, although the bands seemed a bit smeared. Then we did transformation of ccaS_EcoRI, amilGFP_PstI, pSB1A3-amilCP, pSB-pcpcG2 and pSB4A5 backbone. Re-streak (There were one colony on the whole plate) and colony PCR of tetR inverter (DNA taken from well distributed from MIT).

Started overnight cultures of B1001 terminator, PlacO, PSB3T5 and PSB4K5-backbone (prepared for frozen stock + plasmid preparation) and finally RFP with RBS and lambda c1.inv (prepared for frozen stock + plasmid preparation).

The last thing we did was to make new plates of all the three antibiotics (A, C, K). We also made some plate with tetracycline.

2011-07-14

Re-streaking of ccaS_EcoRI, pGEM-amilGFP and pSB4A5. We were supposed to re-streak pSB1A3-amilCP and pSB1A3-PcpcG2 as well, but the transformants had no colonies at all. We suspect wrong plasmid backbone or no ligase added during the cloning attempt. To investigate this the transformation was redone and we re-cloned pSB1A3-amilCP and pSB1A3-PcpcG2 again. O/n of pSB1A3 from the strain collection to test whether we had the right backbone in the frozen stock. We also transformed pSB1A3 backbone, and plated the transformants on A, C and K-plates. We have to find out what went wrong with the transformation of pSB1A3-amilCP and pSB1A3-PcpcG2. Plasmid prep of pSB1AK3-B1001, pSB1A2-R0011, pSB3T5-backbone, pSB4K5 backbone. Freezing glycerol stocks of pSB4K5-B0034 and λ-c1.inv. PCR amplification of tetR.inv with phusion, ran a gel of the PCR products.

The Chromophore The second Biobrick assembly step on the chromophores was performed. Parts assembled: B0034-pcyA + B1001 and PLlacO (R0011) + B0034-ho1.

Output/promoter test plasmid Tomas extracted the standard promoter J23101, pSB3K3 and EYFP (E0034) from the MIT kit in order to prepare the test plasmid.

Evaluation The sequencing result arrived today around lunchtime and they were really good. Both clones of B0034-pcyA that we sent looked fine. B0034-ho1 also looked good. The only clone sent of ccaR looked good. ccaS from Tabor’s plasmid was correctly amplified.

Summary so far

Things are going as planned but we have encountered some problem. We have successfully performed the first BioBrick assembly on the chromophore, extracted ccaR, ccaS and PcpcG2 from pJT122, mutagenized amilGFP_EcoRI and amilGFP_EcoRI and prepared a frozen stock of all the constructs generated so far. The sequencing data confirmed that everything was in order. However, we are still working on cph8 and have to incorporate new inverters in the system. We are happy with what we have accomplished so far and excited to tackle the challenges ahead.

2011-07-15

Erik L cloned tetR inverter into pSB1K3 and later transformed it. Lidaw and Johanna continued with the transformation of B0034-pcyA-B1001, Laura and Sibel transformed PLlacO-B0034-ho1 and pSB4K5. We did plasmid preparation of pSB1K3-B0032, o/n of pSB1C3-CcaS_EcoRI, pGEM-amilCP_PstI and pBS4A5.

mCherry is an alternative red output module that we found in the BioBrick registry. We decided to try it out since its red output is stronger than RFP. Thus we transformed the mCherry (J06702, with RBS and double terminator) from the kit.

2011-07-16

Started by doing plasmid preparation and freezing PSB4A5 stock. We also did plasmid preparation of pSB1A2-B0034. Then we proceeded to screen of PcpcG2 and pGEM-amilCP_PstI.

Made glycerol stock and & mutagenesis/BB10 PCR of pSB1C3-CcaS_EcoRI (-->ccaS_EcoRI_SpeI), pGEM-amilCP_PstI and pBS4A5 (amilGFP_Pst1_BB10). Only amilGFP_Pst1_BB10 showed a band on the gel. Re-streak of the transformations from yesterday and o/n of pcpcG2 and amilCP.

2011-07-17

Frozen stock and plasmid preparation of pcpcG2 and amilCP. Phosphorylation (Phosphorylation protocol), PCR purification, Diegestion, re-ligation, transformation and plating of ccaS_EcoRI_SpeI. PCR of AmilGFP_BB10. Plasmid preparation of pcpcG2 and amilCP.

O/n culture of: pSB3T5_CA2A (B0034-pcyA-term) pSB4K5_CA2B (pLlacO-B0034-ho1) pSB1K3_tetR.inv pSB1A3_mCherry pSB3K3-backbone pSB1K3_YF1 pSB1C3_BFP J61002-J23101 (test plasmid promotor)

Week 5

2011-07-18

Ran CA2A (B0034-pcyA-term), CA2B (pLlacO-B0034-ho1) and tetR.inv on gel and plasmid prepared of all the o/n cultueres from yesterday. Phusion PCR amplification (using BB10 primers) of non-Biobrick compatible cph8 from Voigt’s plasmid. This attempt was successful. We finally managed to get cph8. Gel ran for TagBFP and amilGFP. TagBFP came from colony PCR, and they turned out to have the right length.

Chromophore We finally put together the final step in the chromophore assembly; CA2B + CA2A --> CA3

Red output assembly PompC + tetR.inv

Green output assembly PcpcG2 + B0034

Blue output assembly B0034+ amilCP

Green sensor assembly B0034 + ccaR B0034 + ccaS_EcoRI_SpeI (completely mutated, BioBrick-compatible)

We also assembled J23101 + mCherry (J06702)

Frozen stock pSB3T5_CA2A pSB4K5_CA2B pSB1K3_tetR.inv J61002-J23101– test plasmid pSB1A3_mCherry pSB3K3-backbone pSB1K3_YF1

λ Red Create new plasmid for Lambda Red…………

YF1, FixJ and FixK2 promoter (or PfixK) arrived today. Due to the overwhelming amount of work, we decided to wait for a couple of days before transforming them.

2011-07-19

Transformation of all the Biobrick assemblies from yesterday and performed colony PCR on the plated ccaS_EcoRI_SpeI from 2011-07-17. We also did some O/N cultures of ccaS_EcoRI_SpeI. The gel showed nice and clear bands of the appropriate length.

Cloning of amilGFP_BB10 into pSB1K3 and later it was transformed together with YF1, FixJ and PfixK. These are parts of the blue sensor and they were synthesized by GenScript. Cph8 was cloned into pSB1K3.

The following parts were sent to sequencing: pcpcG2, CcaS_EcoRI_SpeI, CA2A, CA2B and tetR.inv. TagBFP and amilCP are sequenced by Erik Gullberg via IMBIM.

2011-07-20

Plasmid preparation of B0034-ccaS_EcoRI_SpeI, Re-streaking of pSB4A4-CA3, pSB1C3-PompC-tetR.inv, pSB1K3-B0034-ccaR, pSB1C3-B0034-amilCP, pSB1C3-PcpcG2-B0034, pSB1A3-J23101-mCherry. Transformation of pSB1K3-cph8 and Re-streak of pSB1K3-amilGFP, YF1, fixJ and PfixK. This was done late because they were transformed very late. By the end of the day we did new ampecilin plates since there were non left.

The lambda red assembly plan must gain higher priority. The first assembly line involving EnvZ-KO could’ve been started earlier, but hasn’t. Thus, we have to hurry up the work on λ Red.

2011-07-21

Screening and o/n culture of the re-streaked plate from yesterday. Followed by Re-streak of pSB1K3-cph8, pSB1A3-B0032-BFP, pSB1K3-B0034-BFP, pSB1C3-B0032-YF1, pSB1C3-B0034-YF1. We plasmid prepared pSB1K3 and pSB1C3 backbones.

Erik L became responsible for λ-Red in order to keep up with the assembly. He takes guidance from Erik Gullberg (supervisor) and together they lay down a good approach. Evaluation

Sequencing result of ccaS showed an unwanted insert. It’s probably because of bad primer alignment during mutagenesis. Our initial conclusion was that the primers amplified the DNA of some contaminants. Theoretically, this shouldn’t have occurred, since we did PCR purification. The small DNA that caused the insert should’ve been removed.

Erik Gullberg thinks we might have taken a bad clone from the plate when doing mutagenesis to remove EcoRI. So he thinks we should take another clone post-EcoRI and tries it out again. The result of the sequencing reflects a very rare accident. Theoretically we should be able to do it again, without having the same problem. Otherwise, we are considering having Bioneer synthesize ccaS with codon optimization. CA2B samples were gave poor results, it seemed like we accidently put two primers into the sequencing sample. This is probably due to stress.

Because of the work intensity and the stress people are starting to feel right now. Suggestions came up that we should move our weekly meeting on Mondays to Friday afternoon. Considering the current situation we decided to try it out next week.

2011-07-22

Frozen stock of: amilGFP YF1 FixJ PfixK pcpcG2-B0034 pompc-tetR.inv

PCR purification of B0034-ccaR and the above mentioned parts. New Re-streaking of 8 colonies of ccaS_EcoRI. This is because we believe we were unlucky to pick a colony with insert. Thus we try our luck with other colonies. Then we plasmid prepared amilGFP, B0034-ccaR, pcpcG2-B0034 and pompc-tetR.inv.

Cloning of: pSB1AK3-YF1 pSB1C3 pUC57-fixJ pSB1K3 pUC57-YF1 pSB1K3 pUC57-PfixJ pSB1K3

O/N cultures and PCR screening of B0032-BFP, B0034-BFP, B0034-YFP and B0032-BFP.These are the parts of the output/promotor test plasmid assembly.

2011-07-23

Screening of : pSB4A5-CA3, pSB1C3-ccaS_EcoRI and pSB1C3-B0034-amilCP. Followed by screening of pSB1C3 – CcaS_EcoRI_SpeI (has insert).

We also made screening of Cph8, 4 samples (VF2 and VR, biobrick backbone) and making the glycerol frozen stock of Chp8 (un-mutated, Biobrick Backbone). Screened with gel and all the bands had accurate lengths.

Glycerol frozen stock & plasmid prep of: pSB1A3-B0032-BFP pSB1K3-B0034-BFP pSB1C3-B0032-YFP pSB1C3-B0034-YFP Mutagenesis of pSB1C3 – CcaS_EcoRI_SpeI (new 2nd mut).

Output/promotor test plasmid assembly 3A assembly of: PompC-tetR.inv + B0034-BFP PompC-tetR.inv + B0034-RFP PompC-tetR.inv + B0034-RFP

Green output assembly pSB1A3 – PcpcG2 + B0034-amilGFP

Last thing was making O/N culture of pSB1C3-B0034-amilCP and of CA3.

Evaluation The new Ccas-EcoRI showed good bands, Ccas_EcoRI_SpeI did not (as expected due to illegal insert of approximately 50 bp shown after sequence data). The reason why we screened it again was to compare the band length with the new mutation of Ccas-EcoRI. Cph8 insert was successfully cloned in the pSB1K3. We had to redo PCR procedure of B0034-amilCP and do the second, new mutagenesis of ccaS_EcoRI to remove illegale SpeI restriction site.

2011-07-24

Started by running a gel on PCR-amplification products from yesterday. O/N- culture of pSB1C3-B0034-amilCP from yesterday had to be redone. The negative control was contaminated. CA3 was plasmid prepared.

3A assembly followed by transformation of

Output/promotor test plasmid assembly pSB1A3-J23101 + B0032-BFP pSB1K3-J23101 + B0032-YFP pSB1A3-PcpcG2 + B0034-BFP pSB1K3-PcpcG2 + B0034-YFP

Inverter test plasmid assembly pSB2K3-I0500 (Para/BAD) pSB1A2-E0430 (YFP)

Additional transformation of: pSB1K3-YF1 pSB1K3-FixJ pSB1K3-PfixK pSB1K3 -PompC-tetR.inv-B0034-BFP pSB1K3- PompC-tetR.inv-B0034 RFP pSB1K3- PompC-tetR.inv-B0034-YFP

Evaluation ccaS_EcoRI_SpeI (new 2nd mutagenesis) showed no bands which means it need to be redone. B0034 – amilCP has accurate length. Lei tried to improve the result by change the annealing temperature to 57,3 oC. Cph8 showed accurate length.

Week 6

2011-07-25

Re-streak and screening of all transformants from yesterday. New transformation of pSB2K3-I0500 due to no colonies from yesterdays plating.

Making Lambda Red compatible BioBrick plasmids Screening of BioBrick plasmid pSB3T5_specR by using designed primers PEG3x5F (containing the whole terminator part and SacI restriction site) and primer, PEG3x5F2 (containing a part of the terminator and without SacI restriction site)

O/N-culture of pSB4A5-CA3, pSB1C3-B0034-amilCP and pSB1C3-YFP. Lei used his new protocol to screen ccaS_EcoRI_SpeI. He proceeded with phosphorylation, re-ligation and transformation.

Evaluation specR showed no band for the primer PEG3x5F. We decided to do PCR again by using primer PEG3x5F2 as a template, PEG3x5F and PEG3x5R as primers in order to check the functionality of the primers PEG3x5F2. The new result was to short bands on the gel. The primer PEG3x5F2 doesn't work and we need to design and order new primers. The mutagensis product of ccaS_EcoRI_SpeI showed accurate length on the gel and was therefore re-circulized.

2011-07-26

Re-streak of pSB2K3-I0500 and CcaS_EcoRI_SpeI. Second screening of pSB1K3-CcaSEcoRI_SpeI to ensure we got the cells with correct insert. This was followed by PCR purification.

PCR of pSB1A2-PcpcG2-B0034-amilGFP.

Plasmid preparation and glycerol-stock of: pSB1C3-E0034 (eYFP) pSB4A5-CA3 pSB1C3-B0034-amilCP

O/N-culture of re-streaked colonies: pSB1K3-YFI pSB1K3-FixJ pSB1K3-PfixK pSB1K3-cph8 pSB1C3-CcaSEcoRI_SpeI pSB1A2-E0430 (YFP)- test plasmid pSB1A3-pcpcG2-B0034-amilGFP

Prepared samples for sequencing (CA3, B0034-amilCP, cph8, ccaS_EcoRI_SpeI, pcpcG2-B0034, PompC-tetR.inv, B0034-CcaR, pcpcG2-B0034-amilGFP).

Evaluation The screening of CcaS_EcoRI_SpeI showed accurate length on the gel and was sent for sequencing. PompC-tetR.inv-B0034-RFP showed wrong length.

2011-07-27

Yesterdays’ samples were sent for sequencing. Glycerol stock and plasmid prep of the O/N cultures from Yesterdays’. Mohammed and Pikkei made new LB medium. We also screened our yellow output (PcpcG2-B0034-amilGFP) and the band on the gel showed accurate length.

Red output assembly PompC-tetR.inv + B0034-RFP in pSB1K3-B0032 and pSB1K3-B0034-RFP. We wanted to the test the output since the output can become either red or colorless.

O/N -cultures of pSB2K3-I0500 (inverter test plasmid assembly).

Evaluation The screening showed that the assembly of yellow output was done successfully.

2011-07-28

Today we did plasmid prep and glycerol stock of pSB2K3-I0500. Re-sreak of transformed and plated λ-Red plasmid and envZ amplicon (contains Kan-cassette)

Screening of: YFI FixJ PfixK PcpcG2-B0034-BFP PcpcG2-B0034-YFP J23101-B0032-BFP J23101-B0032-YFP PompC-tetR.inv-B0034-BFP PompC-tetR.inv-B0034-YFP

O/N cultures of pSB1A3-PcpcG2-B0034-BFP, pSB3K3-J23101-B0032-BFP pSB3K3-J23101-B0032-YFP and pSB1K3-PcpcG2-B0034-YFP.

Evaluation The band on the gel for the screening showed correct length of all samples except for the PompC-tetR.inv-B0034-BFP.

2011-07-29

Johanna and Laura did glycerol stock and plasmid prep of the O/N - cultures from 2011-07-28. They also re-transformed the red output (PompC-tetR.inv + B0034-RFP) and did the experiment from 2011-07-27 again since we could come to a solid conclusion the first time.

Blue output assembly PfixK+λC1.inv

Blue sensor assembly B0034+FixJ B0034+YF1 These assemblies were later transformed.

O/N- cultures of pSB1K3-PcpcG2-B0034-YFP

Evaluation There were no colonies on the re-streaked plate from yesterday. The result of the red output-assembly showed a plate with only white colonies (contain backbone pSB1K3-B0032) and one with both red and white (contain backbone pSB1K3-B0034-RFP). We picked the white colonies from the plate where the insert was put in pSB1K3-B0034-RFP- backbone which from now on be called red output with RFP (ROR). Hopefully this contains our insert and does not become induced by the TOP10-strain.

2011-07-30

Re-streak of all yesterday’s transformants. Today we also started making new competent TOP10 cells and inoculated them for the night. λ-Red plasmid is still processing slowly. But we ran a PCR to amplify envZ-KAN gene (Kan-cassette), followed by screening. O/N culture and screening of pSB1K3-PompC-tetR.inv-B0034-RFP. Point mutagenisis and O/N on pSB1K3-cph8_PstI since earlier mutagensis (first on) didn't work. Re-pleated, amplified and screening of earlier mutaded pSB1K3-Cph8_PstI.

Evaluation The gel picture of amplified pSB1K3-cph8_PstI showed good result. It also seemed like the point mutagenesis of pSB1K3-cph8_PstI had an accurate length. The reason why we did a new mutagenesis is because we had a lot of trouble with our sensor before and we wanted to be on the safe side. The gel picture of envZ-KO and the red output didn't show any band and needs to be redone. At this stage the failure rate has increased leading reforms in the standard procedures so far. However, we are coming closer to the goal and need to keep to going.

2011-07-31

Testing the new top 10 competent cell by doing a transformation of PUC57 (with PfixK gene) and plating it on Amp plate. Running a gel of PfixK-λC1, B0034-YF1 and B0034-FixJ.

(egentligen 1/8, men jag flytta för att det ska passa med summary) Colony PCR of B0034 - YF1 and B0034 – FixJ followed by screening. Additional screening of ROR and envZ-KO.

PT5lac promotor arrived today

Summary so far

Blue output: B0034-amilCP (BOA) is assembled and sequenced and is ready for the final step. PfixK-λC1.inv is assembled but not yet sequenced. Blue output will soon be ready for the final assembly.

Red output: Final assembly has been done several times with unsuccessful result. The reason for the problem is not clear. There seems to be a problem in the assembly of PompC-tetR.inv + B0034-RFP. Over and over again we can only see the band for tetR.inv on the gel. The sequence comes back with bad result every time. However, sequencing of the two individual components is good. Our screening method is might not be good enough.

We decided to resume the attempt with mcherry and use it as a backup in case we would fail totally.

Green output: Final assembly (pSB1K3-pcpcG2 + B0034-amilGFP) is done and the sequencing data showed perfect assembly. However, we are considering putting the final construct in a low copy plasmid (pSB3C5) because the promotor is slightly leaky.

Chromophore assembly: Last assembly pSB4A5-PlacO-B0034-ho1 (CA2B) + B0034-pcyA-Term (CA2A) has been done but the sequencing result showed double peaks. Needs to be sequenced again. We will also check the previous assembly step CA2B by sending it for sequencing. At this stage we are considering an alternative assembly. We believe that ho1 activity decreases the concentration of Hem. Hence we will put the PLlacO promotor last.

Red sensor: chp8 has been mutagenized two times to remove the illegal PstI site. The first time showed a large insert. Thus the mutagenezis has to be redone and send for sequencing.

Green sensor: the mutation of ccaS has been a tough one. It has to be mutagenized two times and the first sequencing result showed an insert. Assuming that we were un-fortuned to pick a colony with inserts. We went back and choose other colonies. It was the right assumption because the second sequencing was correct. ccaR is already assembled with B0034.

Blue sensor: Since the arrival of all the components the assembly of the blue sensor took of quit well. B0034-fixJ showed good sequencing results but B0034-YF1 showed no bands. Thus it needs to be assembled again!

λ-red: Is lagging behind and has to be taken care of. All the attempts with envZ-KO so fare show modest or no results.

Modelling: remains one of the toughest challenges. So far we haven’t found a model that can be easily implemented and understood. We are still working on it.

Bioneer We sent an order to one of our sponsors Bioneer to synthesize tree color proteins that we can use as alternative output modules. aeBlue and cjBlue are different tones of blue and eforRed is a red protein.

Test plasmid assembly Antionio and Thomas are focusing on making test plasmids. The aim to be able to meassure the output, promotor activity as well as inverter activity.

Week 7

2011-08-01

Glycerol stock and plasmid preparation of PfixK-λC1.inv and red output. The chromophore (CA2A + CA2B) was PCR amplified together with B0034-RFP. Transformation of BBa_I732731.

2011-08-02

PCR purification of B0034-FixJ followed by plasmid preparation and freezing. Transformation of PT5lac. PCR purification of yesterday and preparation for sequencing. Re-streak of BBa_I732731 and screening of envZ-KO and B0034-YF1. O/N-culture of BBa_I732731.

O/N-culture of pSB3C5, pSB4C5 and pSB1C5

Green sensor assembly B0034 + ccaS pSB1K3

2011-08-03

Plasmid preparation and glycerol stock of yesterday’s O/N-culture and cloning of pSB1K3-pcpcG2-B0034-amilGFP pSB3C5. Re-streak of PT5lac and O/N culture of pSB4A5-CA3, pSB3K5, and PompC-tetR.inv.

Output/promotor test plasmid assembly PompC-tetR.inv+B0034-BFP PompC-tetR.inv+B0034-YFP

Blue output assembly PfixK+λC1.inv + B0034-amilCP

Blue sensor assembly B0034 + YF1

Sent for sequencing: CA2A, CA2B, PompC-tetR.inv, B0034-RFP, chp8_ PstI (new mut), B0034-FixJ, B0034-YF1.

2011-08-04

Transformation of chp8_ PstI, pcpcG2-B0034-amilGFP, plasmid preparation and glycerol stock of BBa_I732731, PompC-tetR.inv and CA3. Re-streak of blue sensor and output (B0034-YF1, PfixK- λC1-B0034-amilCP), PompC-tetR.inv-B0034-BFP and of PompC-tetR.inv-B0034-YFP. Screening of B0034-ccaS. Digestion of pSB1C3-B0034-YF1 with XbaI and with pstI- restriction enzyme. PCR amplification of CA3, screening and O/N-culture of PT5lac.

Output/promotor test plasmid assembly PfixK- λC1.inv + B0034-YFP PfixK- λC1.inv + B0034-BFP

Inverter test plasmid assembly Para/BAD + B0034-BFP Para/BAD + B0032-BFP

Green sensor assembly B0034+ccaS pSB1K3

Red sensor assembly B0034+cph8 pSB1C3

O/N- culture of PompC-tetR.inv, PompC-tetR.inv-B0034-RFP, B0034-ccaS

Evaluation B0034-ccaS and PT5lac showed accurate length on the gel. Digestion of pSB1C3-B0034-YF1 because we wanted to check the length of the insert. Furthermore the sequencing result were good thus we can proceed with the following steps.

2011-08-05

Started with transformation of Para/BAD-B0034-BFP, Para/BAD-B0032-BFP, B0034-ccaS, B0034-cph8. We also did Plasmid prep and glycerol stock of PompC-tetR.inv-B0034-RFP, B0034-ccaS.

We ran B0034-YF1, envZ-KO and CA3, on a gel and all the samples showed accurate length this time. PCR purification of B0034-ccaS.

Screening of: pSB1A3-PompC-tetRinv-B0034-RFP pSB1A3-PompC-tetRinv-B0034-YFP

Output/promotor test plasmid assembly PfixK- λC1.inv + B0034-YFP PfixK- λC1.inv + B0034-BFP

Re-streak of chp8_ PstI. O/N- culture of B0034-YF1, PfixK- λC1-B0034-amilCP.

Lambda red plasmid (Anto. pikkei…..)

Evaluation No band shown after gel screening of pSB1A3-PompC-tetRinv-B0034-RFP and pSB1A3-PompC-tetRinv-B0034-YFP thus no need for O/N- Culture yet. The screening was redone with a lower annealing temperature.

2011-08-06

Re-streak of yesterday’s transformats. Successful screening of pSB1A3-PompC-tetRinv-B0034-BFP and pSB1A3-PompC-tetRinv-B0034-RFP. Hence, we proceeded with O/N-culture B0034-YF1 and PT5lac.

Screening of; PfixK-λC1.inv-B0034-amilCP B0034-YF1 PUC57-PT5lac

Evaluation Only B0034-YF1 gave good result. New blue assembly of blue output is required.

Green sensor assembly B0034-ccaS + B0034-ccaR pSB4C5 Followed by transformation

2011-08-07

Amplification of λ-red plasmid and PCR purification of envZ-KO and B0034-YF1. We also did plasmid preparation and glycerol stock of yesterday’s O/N-cultures and re-streak of B0034-ccaS-B0034-ccaR. We made new agar plates, and as usually plates with A, C and K resistance.

Screening of: B0034-ccaS PfixK-λC1.inv-B0034-YFP PfixK-λC1.inv-B0034-BFP B0034-cph8 PcpcG2-B0034-amilGFP PcpcG2-B0034-BFP J23101-B0032-BFP PcpcG2-B0034-YFP J23101-B0032-YFP

Evaluation Good results for: PcpcG2-B0034-BFP J23101-B0032-BFP J23101-B0032-YFP PcpcG2-B0034-YFP PcpcG2-B0034-amilGFP B0034-ccaS

Bad result for: PfixK-λC1.inv-B0034-YFP PfixK-λC1.inv-B0034-BFP B0034-cph8

Week 8

2011-08-08

PCR purification of yesterday’s screens followed by glycerol and plasmid. Cloning of PT5lac pSB1C3 and O/N-culture of Para/BAD-B0032-BFP and B0034-ccaS-B0034-ccaR.

Output/promotor test plasmid assembly J23101 + B0034-BFP J23101 + B0034-YFP J23101 + B0034-amilCP J23101 + B0034-amilGFP

2011-08-09

Transformation of pSB3C5, pSB3K5 and pSB3S5 for later λ-red constructs. Additional transformation J231001-B0034-amilCP and J231001-B0034-amilGFP. The assembly of J23101 + B0034-BFP and J23101 + B0034-YFP didn’t succeed. Plasmid preparation of Para/BAD-B0032-BFP and B0034-ccaS-B0034-ccaR.

Screening of: PfixK-λC1.inv-B0034-YFP, PfixK-λC1.inv-B0034-BFP and B0034-cph8 again. pSB1A3-PompC-tetRinv-B0034-YFP pSB1A3-PompC-tetRinv-B0034-RFP pSB1A3-PompC-tetRinv-B0034-BFP Para/BAD-B0032-BFP Para/BAD-B0034-BFP B0034-ccaS-B0034-ccaR

Evaluation Good results only for: Para/BAD-B0032-BFP B0034-ccaS-B0034-ccaR

New assemblies for the failed screenings

Sent for sequencing: B0034-YF1- B0034-FixJ, PfixK-λc1-inv-B0034-amilCP, PT5lac, PfixK-λc1 inv-B0034-YFP, B0034-ho1-B0034-PcyA-term, RBS-cph8, B0014-PlacO, Para/Bad-RBS-BFP, pSB3K3-K098010 and pSB1A3-PompC-tetRinv-B0034-RFP

2011-08-10

Re-streak of J231001-B0034-amilCP and J231001-B0034-amilGFP. New assemblies made for failed sequencing followed by transformation.

Blue output assembly New assembly of PfixK-λC1.inv + B0034-amilCP Followed by transformation

Output/promotor test plasmid assembly PfixK-λC1.inv + B0034-BFP PfixK-λC1.inv + B0034-YFP Followed by transformation

2011-08-11

Evaluation The sequence data for Para/BAD+B0032-BFP was hard to interpret. The sequence data of the new assembly of pSB4K5-PLlacO-B0034-ho1, pSB1K3-PompC-tetRinv-B0034-RFP and the old assembly of pSB1K3-PompC-tetRinv-B0034-RFP didn’t show satisfying result. However, B0034-RFP segment is clear. The rest of the samples that we sent for sequencing showed good result.

Re-streak of yesterday’s transformants and pSB3C5-FRT backbone and pSB3S5-FRT.

O/N-cultures of pSB1AK3-B0014 pSB1A2-R0011 pSB1K3-J23101-B0034-amilCP pSB1K3-J23101-B0034-amilGFP

Red sensor assembly New assembly of B0034+cph8 pSB1C3 Followed by transformation

Making Lambda Red compatible BioBrick plasmids Repeat cloning of SpecR and KanR-amplicon

2011-08-12

Re-streak of yesterday’s transformants and of A, C, K-backbone.

Screening of: PT5lac PfixK-λC1.inv-B0034-amilCP PfixK-λC1.inv-B0034-YFP PfixK-λC1.inv + B0034-BFP Plasmid prep and frozen stock of: PT5lac PfixK-λC1.inv-B0034-amilCP PfixK-λC1.inv-B0034-YFP pompC-tetR.inv-B0034-RFP J23101-B0034-amilGFP J23101-B0034-amilCP

Assembly of our coupled solution for chromphore and sensor B0014+PLlacO pSB1C3 Output/promotor test plasmid assembly New assembly of PfixK-λC1.inv+B0034-BFP

Blue sensor assembly RBS-YF1+RBS-fixJ Followed by transformation

Chromophore assembly RBS-ho1+RBS-pcyA-B1001 (term) Followed by transformation

Evaluation Since the band of PfixK-λC1.inv + B0034-BFP showed wrong length we need to do the assembly of again. The rest of the samples showed the right length.

Making Lambda Red compatible BioBrick plasmids Ligation of Spec and KanR……..

Transformation of: pSB1A10, the plasmid that are going to be used for the inverter test plasmid. Transformation of different inverter components taken from the BioBrick-kit. Followed by transformation of Lambda red components (SpecR, KanFRT).

2011-08-13

Re-streak of pSB1A10 and RBS-YF1+RBS-fixJ. Additional re-streak of pSB3C5-FRT because of the first attempt failed.

Output/promotor test plasmid assembly J23101 + B0034-BFP (new assembly) J23101 + B0034-YFP (new assembly) PT5lac + B0032-YFP pSB3K3 PT5lac + B0032-BFP pSB3K3 PT5lac + B0034-BFP pSB1K3 PT5lac + B0034-YFP pSB1A3

Red sensor assembly New assembly of B0034+chph8

Inverter test plasmid assembly New assembly of PARA/BAD+B0032 PARA/BAD+B0034 Followed by transformation

Amplification of pfixK-λC1.inv-B0034-amilCP with touch down PCR, since older gel picture showed two bands.

Transformation of: PfixK-λC1.inv-B0034-BFP pSB1C3-B0014-PLlacO pSB3K3-K098010 (Harvard's chromo mod)

O/N-culture of: pSB1A3-backbone pSB1C3-backbone pSB1K3-backbone

Evaluation Still, even though using a touch down approach, we saw one weak second band for the pfixK-λC1.inv-B0034- amilCP amplicon. Next time we will change the protocol with a slightly higher starting and ending temperature.

2011-08-14

O/N culture of pSB3C5-FRT (λ-red plasmid) and screening of RBS-YF1+RBS-fixJ.

Re-streak of PfixK-λC1.inv-B0034-BFP B0014-PLlacO B0034+chph8

Antonio Digest; pSB3C5SpecR pSB3X5Kan-FRT

Plasmid prep and glycerol stock of pSB1A3-backbone pSB1C3-backbone pSB1K3-backbone

Second amplification attempt of PfixK-λC1.inv-B0034-BFP with an alter touch down PCR protocol. Re-streak of yesterday’s transformants.

Transformation of pSB1A2-J06504 (mcherry, from kit) pSB1K3-J23101-B0034-YFP pSB1K3-pT5lac-B0034-BFP pSB1K3-pT5lac-B0034-YFP pSB3K3-pT5lac-B0032-YFP pSB3K3-pT5lac-B0032-BFP

Week 9

2011-08-15

Plasmid prep and glycerol stock of pSB3C5-FRT, RBS-YF1+RBS-fixJ and pSB1A10. Re-streak of yesterday’s transformations.

Screening and O/N of: B0034+chph8 pSB3K3-K098010 B0014-PLlacO PT5lac-B0034-YFP RBS-ho1-RBS-pcyA-B1001

Output/promotor test plasmid assembly pSB1A3-PompC-tetRinv + B0034-RFP Followed by transformation Tomas Transformation and re-streak pSB3C5SpecR pSB3X5Kan-FRT

Evaluation Satisfying screening results. However, pSB3C5-FRT, RBS-YF1+RBS-fixJ failed again. Wrong band on the gel.

2011-08-16

PCR purification of yesterday’s screens

Antonio Digest; pSB3C5-Kan-FRT with Sal1 & Sac1

Ligation of al ready digested components:

Output/promotor test plasmid assembly pSB1K3-PT5lac + B0032-YFP pSB1K3-PT5lac + B0034-BFP pSB1K3-PT5lac + B0034-BFP

2011-08-17

Transformation of: PomPC-tet.R.inv-B0034-amilCP PT5lac-B0034-BFP PT5lac-B0032-YFP PT5lac-B0032-BFP KanR-FRT SpecR

O/N-cultures of: PfixK-λ C1-B0034-BFP B0014 mCherry PT5lac-B0034-YFP J23101-B0034-YFP J23101-B0034-BFP

Red output assembly PompC-tetRinv + B0034-amilCP --> pSB1A3 A test to see if our assembly method to put our red output together should be done in a diffrent way.

Red sensor assembly B0034-Cph8 + B0014 --> pSB3T5 Digestion of B0034-Cph8+B0014-PlacO --> pSB3T5 The promotor that we have been using might be toxic to the cell, therefor we are trying out different approaches.

Colony PCR of pSB1A3-PfixK-λ C1-B0034-BFP

2011-08-18

Glycerol stock of: PfixK-λC1.inv-RBS-BFP mCherry PT5lac-RBS-YFP J23101-RBS-BFP J23101-RBS-YFP

Restreak of: PompC-tetR.inv-RBS-amilCP SpecR KanR-FRT PT5lac-B0032-YFP PT5lac-B0034-BFP PT5lac-B0032-BFP

Transformation of: RBS-Cph8-B0014-PlacO

Ligation of pSB3T5-B0034-Cph8+B0014-PlacO over night.

Red output assembly Assembly of: RBS+mCherry --> pSB3C5 (Alternative for our red output.) RBS-cph8+B0014 --> pSB3T5

2011-08-19

Re-streak of pSB3T5-RBS-cph8-B0014-PlacO

O/N-culture of J23113 and pSB4K5-LacIq (Repressor)

Screening of: PfixK-λC1.inv-RBS-BFP PT5lac-B0032-YFP PT5lac-B0032-BFP

Transformation of : pSB1A3-PompC-tetRinv-B0034-BFP pSB1K3-PompC-tetRinv-B0034-YFP pSB3K5 FRT backbone (for lambda -red) pSB3T5-RBS-cph8-B0014-PLlacO

2011-08-20

Re-streak of: PT5lac-RBS-YF1-RBS-FixJ RBS-cph8-B0014-PlacO PompC-tetR.inv-B0034-BFP PompC-tetR.inv-B0034-YFP RBS-cph8-B0014

Plasmid prep and glycerol stock of: PT5lac-B0032-YFP PT5lac-B0032-BFP laqIq J23113

Red output assembly New assembly of B0034+mCherry --> pSB1C3 since earlier transformation didn't show any colonies.

O/N-cultures of: KanR-FRT SpecR B0034-cph8-B0014-PLacO PompC-tetR.inv-B0034-BFP

Screening of: SpecR KanR-FRT pSB3C5-FRT RBS-cph8-B0014-PLacO PompC-tetR.inv-B0034-BFP PT5lac-B0034-BFP

Evaluation The screening showed accurate lenght of the following samples: KanR-FRT SpecR B0034-cph8-B0014-PLacO PompC-tetR.inv-B0034-BFP

2011-08-21

Transformation of B0034-mCherry

Frozen stock and plasmid prep of: B0034-cph8-B0014-PlacO SpecR KanR-FRT

Week 10

2011-08-22

Another, new transformation of B0034-mCherry, since yesterdays transformation didn't show anything on the plate.

Plating of EnvZ-KO (clone one and two)

Screening of: PompC-tetR.inv-B0034-BFP PompC-tetR.inv-B0034-YFP RBS-cph8-B0014

Evaluation Screening of samples,see above, showed accurate band length for all assemblies except for PompC-tetR.inv-B0034-BFP.

2011-08-23

Samples prepared and sent for sequencing: PompC-tetR.inv-B0034-YFP RBS-cph8-B0014-PlacO RBS-cph8-B0014

Blue sensor assembly J23101+RBS-YF1-RBS-FixJ

Green output assembly J23101+B0034-ccaS-B0034-ccaR --> pSB3K3

Red sensor assembly J23101+B0034-cph8 --> pSB3K3

Re-digestion and ligation of: PfixK-λ C1--> pSB1C3 B0034-BFP --> pSB1K3

Re-ligation of B0034-mCherry --> pSB1C3

2011-08-24

Plasmid prep and glycerol stock of: B0034-CcaS-B0034-CcaR J23113

Transformation of: pSB3K3-J23101-RBS-cph8 psB3C5-J23101-RBS-YF1-RBS-fixJ RBS-mCherry PfixK-λ C1-B0034-BFP J23101-RBS-cph8 J23101-RBS-YF1-RBS-FixJ PT5lac-RBS-YF1-RBS-FixJ pSB1K3-J23101-B0034-CcaS-B0034-CcaR

The Chromopher Assembly of J23113-RBS-ho1-RBS-pcyA-B1001, followed by transformation.

Screening of: B0034-CcaS-B0034-CcaR, RBS-YF1-RBS-fixJ, used to compare the band length with, PT5lac-RBS-YF1-RBS-FixJ

Evaluation Screening showed right band-length of PT5lac-RBS-YF1-RBS-FixJ and of B0034-CcaS-B0034-CcaR.

2011-08-25

Screening of : J23101-B0032-BFP J23101-B0032-YFP PT5lac-B0032-BFP PT5lac-B0032-YFP FRT-lacIq EnvZ-KO

O/n- culture of: PT5lac-B0032-BFP PT5lac-B0032-YFP mCherry PT5lac-RBS-YF1-RBS-FixJ

Re-streak of: J23101-RBS-YF1-RBS-FixJ J23101-RBS-cph8 J23101-B0034-ccaS-B0034-ccaR J23113-RBS-ho1-RBS-pcyA PfixK-λ C1-B0034-BFP

Evaluation Gel picture of screened samples showed good length except for EnvZ-KO.

2011-08-26

Red output assembly Redo assembly of B0034+mCherry --> pSB1C3

Screening of: J23101-RBS-YF1-RBS.FixJ PfixK-λ C1-B0034-BFP J23101-RBS-cph8 FRT-lacIq

O/n-culture of: J23101-RBS-YF1-RBS-FixJ PfixK-λ C1-B0034-BFP J23101-RBS-cph8 FRT-lacIq

Evaluation Very bad gel picture due to the agarose.

2011-08-27

Screening and O/n-culture of: J23101-B0034-ccaS-B0034-ccaR J23113-RBS-ho1-RBS-pcyA-B1001 envZ-KO

Transformation of: B0034-mCherry Pfixk

2011-08-28

Output/promotor test plasmid assembly PcpcG2+B0032-BFP --> pSB3K3 PcpcG2+B0032-YFP --> pSB3K3 PlacO+B0032-BFP --> pSB3K3 PlacO+B0032-YFP --> pSB3K3 J23113+B0032-BFP --> pSB3K3 J23113+B0032-YFP --> pSB3K3

Re-streak of: J23101-B0034-amilCP B0034-ccaS-B0034-ccaR-RBS-cph8-B0014 RBS-ccaS-RBS-ccaR-RBS-cph8-B0034-PlacO PfixK

Week 11

2011-08-29

Screening of: pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014 pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014-PLlacO PfixK

O/n-culture of: J23113-RBS-ho1-RBS-pcyA-B1001 J23101-RBS-cph8 FRT-lacIq J23101-B0034-amilCP B0034-ccaS-B0034-ccaR-B0034-cph8-B0014 B0034-ccaS-B0034-ccaR-B0034-cph8-B0014-PlacO PfixK

Blue backbone assembly J23101-B0034-amilCP+B0034 --> pSB1C3 J23101-B0034-amilCP --> pSB1C3

Red output assembly B0034+mcherry --> pSB1C3

Transformation of: pSB3K3-PcpcG2-B0032-BFP pSB3K3-PcpcG2-B0032-YFP pSB3K3-PLlacO-B0032-BFP pSB3K3-PLlacO-B0032-YFP pSB3K3-J23113-B0032-BFP pSB3K3-J23113-B0032-YFP

Evaluation Couldn't see any band on the gel for PfixK and the bands for samples pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014 and pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014-PLlacO had the wrong length.

2011-08-30

Another screening of pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014 pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014-PLlacO but this time using both taq and phusion polymerase.

Transformation of J23101-B0034-amilCP-B0014 B0034-mcherry J23101-B0034-amilCP

PCR purification of: RBS-cph8 J23113-RBS-ho1-RBS-pcyA-B1001

Re-streak of: pSB3K3-PcpcG2-B0032-BFP pSB3K3-PcpcG2-B0032-YFP pSB3K3-PLlacO-B0032-BFP pSB3K3-PLlacO-B0032-YFP pSB3K3-J23113-B0032-BFP pSB3K3-J23113-B0032-YFP

Output/promotor test plasmid assembly PfixK+B0032-BFP --> pSB3K3 PfixK+B0032-YFP --> pSB3K3

2011-08-31

Screening of: pSB3K3-PcpcG2-B0032-BFP pSB3K3-PcpcG2-B0032-YFP pSB3K3-PLlacO-B0032-BFP pSB3K3-PLlacO-B0032-YFP pSB3K3-J23113-B0032-BFP pSB3K3-J23113-B0032-YFP

2011-09-01

Transformation of: PfixK-B0032-BFP PfixK+B0032-YFP

PCR amplification of FRT-lacIq

2011-09-02

Re-streak of: PfixK-B0032-BFP PfixK-B0032-YFP

2011-09-04

O/n- culture of: J23101-B0032-BFP J23101-B0032-YFP PT5lac-B0032-BFP PT5lac-B0032-YFP PcpcG2-B0032-BFP PcpcG2-B0032-YFP PLlacO-B0032-BFP PLlacO-B0032-YFP J23113-B0032-BFP J23113-B0032-YFP

Week 12

2011-09-05

Screening of: FRT-lacIq Pt5lac-RBS-YF1-RBS-FixJ J23101-RBS-YF1-RBS-FixJ

Evaluation The screened samples showed right length for FRT-lacIq but not for the rest.

2011-09-07

Plasmid prep and frozen stock of: J23101-B0034-amilCP J23113-RBS-ho1-RBS-pcyA-B1001 pGEM-cjBlue pGEM-eforRed J23101-RBS-amilCP-B0014

Transformation of pcpcG2 PompC-tetRinv B0034-CcaS CA2A B0032-BFP amilGFP B0034-ccaR YF1 FixJ RBS-ho1-RBS-pcyA-B1001 RBS-YF1-RBS-fixJ B0034-CcaS-B0034-CcaR

2011-09-08

Assembly of: B0034 + eforRed B0034 + pompC-tetR B0034 + mcherry J23101-B0034 + eforRed J23101-B0034 + cjBlue

Re-streak of yesterday’s transformants.

PCR amplification of: B0034-cph8-B0014-PLlacO J23101-RBS-YF1-RBS-fixJ

2011-09-09

O/n-culture of J23101-RBS-amilCP-B0014

PCR amplification: pcpcG2 CA2A B0032-BFP B0034-BFP YF1 FixJ amilGFP cph8_PstI B0034-CcaS RBS-ho1-RBS-pcyA-B1001

Re-streak of: PompC-tetR-B0034 B0034-ccaR B0034-YF1-B0034-fixJ J23101-B0034-eforRed J23113-RBS-ho1-RBS-pcyA-B1001

2011-09-10

O/N-cultur of pSB1C3-J23101-RBS-amilCP-B0014. Digest of J23101-RBS-YF1-RBS-fixJ, pSB4A5-PT5lac-RBS-YF1-RBS-FixJ.Screening of yesderday’s amplicons

Colony PCR of: J23101-B0034-eforRed J23113-RBS-ho1-RBS-pcyA-B1001 B0034-YF1-B0034-fixJ B0034-ccaR PompC-tetRinv-B0034 PT5lac-RBS-YF1-RBS-FixJ J23101-RBS-YF1-RBS-fixJ

Evaluation The gel showed no bands. Suspect that something might be wrong with the gel. Thus, we did a new batch.

2011-09-11

New screening attempt of yesterday’s PCR. It was difficult to see anything significant from the other bands except from that of the digested blue sensor. PT5lac-B0034-YF1-RBS-FixJ and J23101-B0034-YF1-RBS-fixJ.

Tranformation of PT5lac-B0034-YF1-B0034-FixJ, pSB1K3-PfixK-λ C1-B0034-amilCP and pSB1K3-PfixK-λ C1-B0034-YFP in MG1655 strain.

PCR amplification of PT5lac-B0034-YF1-B0034-FixJ for sequencing.

Week 13

2011-09-12

Cloning of constructs into pSB1C3 to be sent to the registry: amilCP PcpcG2-B0034-BFP J23101-B0032-BFP J23101-B0032-BFP J23101-B0032-YFP PcpcG2-B0034-amilGFP B0034-CcaS PcpcG2-B0034-YFP PfixK-λ C1-B0034-amilCP PT5lac-B0032-BFP PT5lac-B0032-YFP 2011-09-12 Re-streak of: B0034-cjGreen B0034-eforRed PfixK-λ C1-B0034

Prepare agar stab BL-21 E.coli strains to be sent to Bilkent Unam Unversity (Turkey, Ankara)

2011-09-13

Transformation of the parts to be sent to the registry. Re-streak of pSB1K3-J23101-B0034-cjGreen followed by O/N.

PCR and gel of; J23101-B0034-CcaS-B0034-CcaR PfixK-B0032-BFP PfixK-B0032-YFP PT5lac-RBS-YF1-RBS-FixJ J23101-RBS-YF1-RBS-fixJ

Screening showed good results.

2011-09-14

Re-streak of: pSB1K3-PfixK-λ C1-B0034-YFP pSB1K3-PfixK-λ C1-B0034-amilCP pSB1K3-J23101-B0034-CcaS-B0034-CcaR pSB1C3-amilCP pSB1K3-PfixK-λ C1-B0034 pSB1C3-PcpcG2-B0034-BFP pSB1C3-PcpcG2-B0034-YFP pSB1C3-PcpcG2-B0034-amilGFP pSB1C3-B0034-CcaS pSB1C3-PfixK-λ C1-B0034-amilCP pSB1C3-PT5lac-B0032-BFP pSB1C3-PT5lac-B0032-YFP pSB1C3-J23101-B0032-BFP pSB1C3-J23101-B0032-YFP

Assembly of: J23101-B0034 + pGEM-eforRed  pSB1K3

Cloning of: pGEM-eforRed  pSB1C3 pGEM-cjGreen  pSB1C3

2011-09-15

O/N-culture and glycerol stock of yesterday’s re-streaks.

Plasmid prep of: RBS-YF1-RBS-fixJ amilGFP B0034-ccaR B0032-BFP B0032-YFP J23113-B0032-BFP PT5lac-B0032-BFP PT5lac-B0032-YFP PcpcG2-B0032-BFP PcpcG2-B0032-YFP PLlacO-B0032-BFP PLlacO-B0032-YFP J23101-B0032-BFP

Assembly of: J23113 + B0032-YFP J23101 + B0034-YFP

2011-09-16

Re-streak of: cjGreen pSB1C3-eforRed J23101-B0034-cjGreen J23101-B0034-eforRed J23113-B0032-YFP J23101-B0034-YFP (YFP from part registry) Screening of: pcpcG2 B0034-BFP YF1 FixJ cph8_PstI B0034-CcaS PfixK-λ C1-B0034 J23101-B0032-YFP PcpcG2-B0034-YFP PcpcG2-B0034-amilGFP B0034-CcaS PfixK-λ C1-B0034-amilCP cph8_PstI PfixK-λ C1-B0034 pSB1C3-amilCP PcpcG2-B0034-BFP J23101-B0032-BFP J23101-B0032-YFP PcpcG2-B0034-YFP PcpcG2-B0034-amilGFP B0034-CcaS PfixK-λ C1-B0034-amilCP PT5lac-B0032-BFP PT5lac-B0032-YFP amilCP

plasmid prep and O/n-culture of: amilCP PcpcG2-B0034-BFP J23101-B0032-BFP PfixK-λ C1-B0034-amilCP PT5lac-B0032-BFP PT5lac-B0032-YFP

Evaluation Everything that we had screened showed the right length except for YF1 and cph8_PstI.

2011-09-17

Re-streak of: J23101-B0034-YFP (Erik Gullbergs YFP) YFP (Erik Gullbergs YFP)

Blue sensor assembly J23101+RBS-YF1-RBS-fixJ  pSB4C5

Output/promotor test plasmid assembly J23101+B0034-YFP  pSB3K3 (Erik Gullbergs YFP)

O/n-culture of: Pt5lac-RBS-YF1-RBS-fixJ RFP amilGFP amilCP cjGreen

J23101-B0034-cjGreen J23113-B0032-YFP

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+Notebook
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-==Notebook==
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 == Week 1 ==
-This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See protocol SOB- medium, LB medium and [[https://2011.igem.org/Team:Uppsala-Sweden/Project/Protocol#Competent_Cell_Preparation_Procedure | competent cell preparation]]). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C  (See Competent cell preparation final). We received the pTJ122 plasmid carrying the ccaS, ccaR and cph8 genes as well as the PcpcG2 promoter from Christopher A Voigt at University of California San Francisco.
+This week was dedicated to make buffers such as CCMB80 and SOC, SOB and LB medium (See the protocols [https://2011.igem.org/Team:Uppsala-Sweden/Project/Protocol#SOB-medium SOB-medium], [https://2011.igem.org/Team:Uppsala-Sweden/Project/Protocol#LB_.E2.80.93_Medium LB medium] and [https://2011.igem.org/Team:Uppsala-Sweden/Project/Protocol#Competent_Cell_Preparation_Procedure competent cell preparation]). We also prepared selective agar plates with ampicillin, chloramphenicol and kanamycin (See agarplate preparation). Finally we prepared competent TOP10 cells and froze them in -80°C. We received the pTJ122 plasmid carrying the ccaS, ccaR and cph8 genes as well as the PcpcG2 promoter from Christopher A Voigt at University of California San Francisco.
 == Week 2 ==
@@ Line 77: / Line 38: @@
 ======2011-06-27======
-This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for transforming TOP10 competent cells. The result was good, we measured a competence efficiency of 1.7 * 10^8 transformants /ug DNA.
+This week we started with testing the competence in our cells. For this transformation we used one positive control (plasmid pUC19 from New England Biolabs) as well as a negative control without plasmid. For the procedure we followed the protocol for [https://2011.igem.org/Team:Uppsala-Sweden/Project/Protocol#Transforming_TOP10_competent_cells transforming TOP10 competent cells]. The result was good, we measured a competence efficiency of 1.7 * 10^8 transformants /ug DNA.
 <span style="color:blue">Blue</span> / <span style="color:green">green</span> output
@@ Line 90: / Line 51: @@
 <span style="color:blue">Blue</span> / <span style="color:green">green</span> output
-Started overnight cultures of E coli carrying the plasmids pGEM11-amilGFP and pGEM14-amilCP.  We followed protocol "Overnight culture and glycerol stock" for the initial preparations on the output modules.
+Started overnight cultures of E coli carrying the plasmids pGEM11-amilGFP and pGEM14-amilCP.  We followed the protocol for [https://2011.igem.org/Team:Uppsala-Sweden/Project/Protocol#Overnight_culture_and_glycerol_stock overnight culture and glycerol stock] for the initial preparations on the output modules.
 Transformation of BioBrick plasmids (protocol for transforming TOP10 competent cells):<br>
@@ Line 104: / Line 65: @@
 pSB1A2-R0011	(PLlacO, lacI repressable promotor) -–Laura & Pekkie<br>
 pSB2K3-I15010	(cph8 red sensor) – Lei & Sibel<br>
@@ Line 120: / Line 82: @@
 Evaluation: All samples were good. pSB1K3 plates with RFP inserts are less red than pSB1A3 and pSB1C3. Re-streaking of the successful transformations from 2011-07-28. The same people who did the transformation proceeded with re-streaking.
 Cph8 had no colonies at all, neither on the 1x or 10X dilution plate. Apparently there seems to be a problem with cph8. Thus, cph8 (red sensor) had to be redone due to failure in the first attempt.
@@ Line 128: / Line 91: @@
 '''Evaluation:'''
 Most transformations succeeded. However, cI inverter gave very little amount of colonies and cph8 failed again. It’s time to use another approach. We got one single colony on the transformation plates and we will screen that one, but if that colony is wrong will try to run a PCR using the standard VF2 and VR primers using the DNA in the kit as template. If there is any BioBrick plasmid in that well, we should get a PCR product. If this will not work out, we need to either synthesize the gene or to PCR up the gene from the Voigt plasmid pJT122. We will then need to clone it into a BioBrick plasmid and run site-directed mutagenesis on it to eliminate the illegal PstI restriction site. Since cph8 is an important part of our project we have to solve this dilemma.
@@ Line 134: / Line 98: @@
 Started by doing overnight cultures from the re-streaked plates from 11-06-30.
 After that Lei and Sibel sterile filtered 20 % glycerol for making glycerol stocks. All the overnight cultures prepared 11-06-30 were frozen in -80°C.
 ======2011-07-02======
@@ Line 140: / Line 106: @@
 == Week 3 ==
 ======2011-07-04======
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 <span style="color:red">Red sensor</span><br>
 At the end of the day we performed colony PCR to verify the length of the single clone we got of red light sensor (cph8) strain.
@@ Line 205: / Line 171: @@
 Digestion of ccaS and ccaR PCR products and plasmid backbone plasmid pSB1C3 by Tomas and Antonio followed up by cloning and transformation by Erik L (BioBrick Assembly Manual). Lidaw And Johanna transformed the RBS-PcyA assembly.
@@ Line 243: / Line 210: @@
 Transformation of parts Q04400 (TetR inverter) and Q04510 (cI inverter).
@@ Line 260: / Line 228: @@
 The last thing we did was to make new plates of all the three antibiotics (A, C, K). We also made some plate with tetracycline.
+======2011-07-14======
+Re-streaking of ccaS_EcoRI, pGEM-amilGFP and pSB4A5. We were supposed to re-streak pSB1A3-amilCP and pSB1A3-PcpcG2 as well, but the transformants had no colonies at all.  We suspect wrong plasmid backbone or no ligase added during the cloning attempt. To investigate this the transformation was redone and we re-cloned pSB1A3-amilCP and pSB1A3-PcpcG2 again. O/n of pSB1A3 from the strain collection to test whether we had the right backbone in the frozen stock. We also transformed pSB1A3 backbone, and plated the transformants on A, C and K-plates. We have to find out what went wrong with the transformation of pSB1A3-amilCP and pSB1A3-PcpcG2.
+Plasmid prep of pSB1AK3-B1001, pSB1A2-R0011, pSB3T5-backbone, pSB4K5 backbone. Freezing glycerol stocks of pSB4K5-B0034 and λ-c1.inv. PCR amplification of tetR.inv with phusion, ran a gel of the PCR products.
+The Chromophore
+The second Biobrick assembly step on the chromophores was performed. Parts assembled: B0034-pcyA + B1001 and PLlacO (R0011) + B0034-ho1.
+Output/promoter test plasmid
+Tomas extracted the standard promoter J23101, pSB3K3 and EYFP (E0034) from the MIT kit in order to prepare the test plasmid.
+Evaluation
+The sequencing result arrived today around lunchtime and they were really good. Both clones of B0034-pcyA that we sent looked fine. B0034-ho1 also looked good. The only clone sent of ccaR looked good. ccaS from Tabor’s plasmid was correctly amplified.
+Summary so far
+Things are going as planned but we have encountered some problem. We have successfully performed the first BioBrick assembly on the chromophore, extracted ccaR, ccaS and PcpcG2 from pJT122, mutagenized amilGFP_EcoRI and amilGFP_EcoRI and prepared a frozen stock of all the constructs generated so far. The sequencing data confirmed that everything was in order. However, we are still working on cph8 and have to incorporate new inverters in the system. We are happy with what we have accomplished so far and excited to tackle the challenges ahead.
+======2011-07-15======
+Erik L cloned tetR inverter into pSB1K3 and later transformed it. Lidaw and Johanna continued with the transformation of B0034-pcyA-B1001, Laura and Sibel transformed PLlacO-B0034-ho1 and pSB4K5. We did plasmid preparation of pSB1K3-B0032, o/n of pSB1C3-CcaS_EcoRI, pGEM-amilCP_PstI and pBS4A5.
+mCherry is an alternative red output module that we found in the BioBrick registry. We decided to try it out since its red output is stronger than RFP. Thus we transformed the mCherry (J06702, with RBS and double terminator) from the kit.
+======2011-07-16======
+Started by doing plasmid preparation and freezing PSB4A5 stock. We also did plasmid preparation of pSB1A2-B0034. Then we proceeded to screen of PcpcG2 and pGEM-amilCP_PstI.
+Made glycerol stock and & mutagenesis/BB10 PCR of pSB1C3-CcaS_EcoRI (-->ccaS_EcoRI_SpeI), pGEM-amilCP_PstI and pBS4A5 (amilGFP_Pst1_BB10). Only amilGFP_Pst1_BB10 showed a band on the gel. Re-streak of the transformations from yesterday and o/n of pcpcG2 and amilCP.
+======2011-07-17======
+Frozen stock and plasmid preparation of pcpcG2 and amilCP. Phosphorylation (Phosphorylation protocol), PCR purification, Diegestion, re-ligation, transformation and plating of ccaS_EcoRI_SpeI. PCR of AmilGFP_BB10. Plasmid preparation of pcpcG2 and amilCP.
+O/n culture of:
+pSB3T5_CA2A (B0034-pcyA-term)
+pSB4K5_CA2B (pLlacO-B0034-ho1)
+pSB1K3_tetR.inv
+pSB1A3_mCherry
+pSB3K3-backbone
+pSB1K3_YF1
+pSB1C3_BFP
+J61002-J23101 (test plasmid promotor)
+== Week 5 ==
+======2011-07-18======
+Ran CA2A (B0034-pcyA-term), CA2B (pLlacO-B0034-ho1) and tetR.inv on gel and plasmid prepared of all the o/n cultueres from yesterday. Phusion PCR amplification (using BB10 primers) of non-Biobrick compatible cph8 from Voigt’s plasmid. This attempt was successful. We finally managed to get cph8. Gel ran for TagBFP and amilGFP. TagBFP came from colony PCR, and they turned out to have the right length.
+Chromophore
+We finally put together the final step in the chromophore assembly; CA2B + CA2A --> CA3
+Red output assembly
+PompC + tetR.inv
+Green output assembly
+PcpcG2 + B0034
+Blue output assembly
+B0034+ amilCP
+Green sensor assembly
+B0034 + ccaR
+B0034 + ccaS_EcoRI_SpeI (completely mutated, BioBrick-compatible)
+We also assembled J23101 + mCherry (J06702)
+Frozen stock
+pSB3T5_CA2A
+pSB4K5_CA2B
+pSB1K3_tetR.inv
+J61002-J23101– test plasmid
+pSB1A3_mCherry
+pSB3K3-backbone
+pSB1K3_YF1
+λ Red
+Create new plasmid for Lambda Red…………
+YF1, FixJ and FixK2 promoter (or PfixK) arrived today. Due to the overwhelming amount of work, we decided to wait for a couple of days before transforming them.
+======2011-07-19======
+Transformation of all the Biobrick assemblies from yesterday and performed colony PCR on the plated ccaS_EcoRI_SpeI from 2011-07-17. We also did some O/N cultures of ccaS_EcoRI_SpeI. The gel showed nice and clear bands of the appropriate length.
+Cloning of amilGFP_BB10 into pSB1K3 and later it was transformed together with YF1, FixJ and PfixK. These are parts of the blue sensor and they were synthesized by GenScript. Cph8 was cloned into pSB1K3.
+The following parts were sent to sequencing: pcpcG2, CcaS_EcoRI_SpeI, CA2A, CA2B and tetR.inv. TagBFP and amilCP are sequenced by Erik Gullberg via IMBIM.
+======2011-07-20======
+Plasmid preparation of B0034-ccaS_EcoRI_SpeI, Re-streaking of pSB4A4-CA3, pSB1C3-PompC-tetR.inv, pSB1K3-B0034-ccaR, pSB1C3-B0034-amilCP, pSB1C3-PcpcG2-B0034, pSB1A3-J23101-mCherry. Transformation of pSB1K3-cph8 and Re-streak of pSB1K3-amilGFP, YF1, fixJ and PfixK.  This was done late because they were transformed very late. By the end of the day we did new ampecilin plates since there were non left.
+The lambda red assembly plan must gain higher priority. The first assembly line involving EnvZ-KO could’ve been started earlier, but hasn’t. Thus, we have to hurry up the work on λ Red.
+======2011-07-21======
+Screening and o/n culture of the re-streaked plate from yesterday. Followed by Re-streak of pSB1K3-cph8, pSB1A3-B0032-BFP, pSB1K3-B0034-BFP, pSB1C3-B0032-YF1, pSB1C3-B0034-YF1.  We plasmid prepared pSB1K3 and pSB1C3 backbones.
+Erik L became responsible for λ-Red in order to keep up with the assembly. He takes guidance from Erik Gullberg (supervisor) and together they lay down a good approach.
+Evaluation
+Sequencing result of ccaS showed an unwanted insert. It’s probably because of bad primer alignment during mutagenesis. Our initial conclusion was that the primers amplified the DNA of some contaminants. Theoretically, this shouldn’t have occurred, since we did PCR purification. The small DNA that caused the insert should’ve been removed.
+Erik Gullberg thinks we might have taken a bad clone from the plate when doing mutagenesis to remove EcoRI. So he thinks we should take another clone post-EcoRI and tries it out again. The result of the sequencing reflects a very rare accident. Theoretically we should be able to do it again, without having the same problem.
+Otherwise, we are considering having Bioneer synthesize ccaS with codon optimization.
+CA2B samples were gave poor results, it seemed like we accidently put two primers into the sequencing sample. This is probably due to stress.
+Because of the work intensity and the stress people are starting to feel right now. Suggestions came up that we should move our weekly meeting on Mondays to Friday afternoon. Considering the current situation we decided to try it out next week.
+======2011-07-22======
+Frozen stock of:
+amilGFP
+YF1
+FixJ
+PfixK
+pcpcG2-B0034
+pompc-tetR.inv
+PCR purification of B0034-ccaR and the above mentioned parts. New Re-streaking of 8 colonies of ccaS_EcoRI. This is because we believe we were unlucky to pick a colony with insert. Thus we try our luck with other colonies. Then we plasmid prepared  amilGFP, B0034-ccaR, pcpcG2-B0034 and pompc-tetR.inv.
+Cloning of:
+pSB1AK3-YF1  pSB1C3
+pUC57-fixJ pSB1K3
+pUC57-YF1 pSB1K3
+pUC57-PfixJ pSB1K3
+O/N cultures and PCR screening of B0032-BFP, B0034-BFP, B0034-YFP and B0032-BFP.These are the parts of the output/promotor test plasmid assembly.
+======2011-07-23======
+Screening of :
+pSB4A5-CA3, pSB1C3-ccaS_EcoRI and pSB1C3-B0034-amilCP. Followed by screening of pSB1C3 – CcaS_EcoRI_SpeI (has insert).
+We also made screening of Cph8, 4 samples (VF2 and VR, biobrick backbone) and making the glycerol frozen stock of Chp8 (un-mutated, Biobrick Backbone). Screened with gel and all the bands had accurate lengths.
+Glycerol frozen stock & plasmid prep of:
+pSB1A3-B0032-BFP
+pSB1K3-B0034-BFP
+pSB1C3-B0032-YFP
+pSB1C3-B0034-YFP
+Mutagenesis of pSB1C3 – CcaS_EcoRI_SpeI (new 2nd mut).
+Output/promotor test plasmid assembly
+A assembly of:
+PompC-tetR.inv + B0034-BFP
+PompC-tetR.inv + B0034-RFP
+PompC-tetR.inv + B0034-RFP
+Green output assembly
+pSB1A3 – PcpcG2 + B0034-amilGFP
+Last thing was making O/N culture of pSB1C3-B0034-amilCP and of CA3.
+Evaluation
+The new Ccas-EcoRI showed good bands, Ccas_EcoRI_SpeI did not (as expected due to illegal insert of approximately 50 bp shown after sequence data). The reason why we screened it again was to compare the band length with the new mutation of Ccas-EcoRI. Cph8 insert was successfully cloned in the pSB1K3. We had to redo PCR procedure of B0034-amilCP and do the second, new mutagenesis of ccaS_EcoRI to remove illegale SpeI restriction site.
+======2011-07-24======
+Started by running a gel on PCR-amplification products from yesterday. O/N- culture of pSB1C3-B0034-amilCP from yesterday had to be redone. The negative control was contaminated. CA3 was plasmid prepared.
+A assembly followed by transformation of
+Output/promotor test plasmid assembly
+pSB1A3-J23101 + B0032-BFP
+pSB1K3-J23101 + B0032-YFP
+pSB1A3-PcpcG2 + B0034-BFP
+pSB1K3-PcpcG2 + B0034-YFP
+Inverter test plasmid assembly
+pSB2K3-I0500 (Para/BAD)
+pSB1A2-E0430 (YFP)
+Additional transformation of:
+pSB1K3-YF1
+pSB1K3-FixJ
+pSB1K3-PfixK
+pSB1K3 -PompC-tetR.inv-B0034-BFP
+pSB1K3- PompC-tetR.inv-B0034 RFP
+pSB1K3- PompC-tetR.inv-B0034-YFP
+Evaluation
+ccaS_EcoRI_SpeI (new 2nd mutagenesis) showed no bands which means it need to be redone. B0034 – amilCP has accurate length. Lei tried to improve the result by change the annealing temperature to 57,3 oC. Cph8 showed accurate length.
+== Week 6 ==
+======2011-07-25======
+Re-streak and screening of all transformants from yesterday. New transformation of pSB2K3-I0500 due to no colonies from yesterdays plating.
+Making Lambda Red compatible BioBrick plasmids
+Screening of BioBrick plasmid pSB3T5_specR by using designed primers PEG3x5F (containing the whole terminator part and SacI restriction site) and primer, PEG3x5F2 (containing a part of the terminator and without SacI restriction site)
+O/N-culture of pSB4A5-CA3, pSB1C3-B0034-amilCP and pSB1C3-YFP. Lei used his new protocol to screen ccaS_EcoRI_SpeI. He proceeded with phosphorylation, re-ligation and transformation.
+Evaluation
+specR showed no band for the primer PEG3x5F. We decided to do PCR again by using primer PEG3x5F2 as a template, PEG3x5F and PEG3x5R as primers in order to check the functionality of the primers PEG3x5F2. The new result was to short bands on the gel. The primer PEG3x5F2 doesn't work and we need to design and order new primers.
+The mutagensis product of ccaS_EcoRI_SpeI showed accurate length on the gel and was therefore re-circulized.
+======2011-07-26======
+Re-streak of pSB2K3-I0500 and CcaS_EcoRI_SpeI. Second screening of pSB1K3-CcaSEcoRI_SpeI to ensure we got the cells with correct insert. This was followed by PCR purification.
+PCR of pSB1A2-PcpcG2-B0034-amilGFP.
+Plasmid preparation and glycerol-stock of:
+pSB1C3-E0034 (eYFP)
+pSB4A5-CA3
+pSB1C3-B0034-amilCP
+O/N-culture of re-streaked colonies:
+pSB1K3-YFI
+pSB1K3-FixJ
+pSB1K3-PfixK
+pSB1K3-cph8
+pSB1C3-CcaSEcoRI_SpeI
+pSB1A2-E0430 (YFP)- test plasmid
+pSB1A3-pcpcG2-B0034-amilGFP
+Prepared samples for sequencing (CA3, B0034-amilCP, cph8, ccaS_EcoRI_SpeI, pcpcG2-B0034, PompC-tetR.inv, B0034-CcaR, pcpcG2-B0034-amilGFP).
+Evaluation
+The screening of CcaS_EcoRI_SpeI showed accurate length on the gel and was sent for sequencing. PompC-tetR.inv-B0034-RFP showed wrong length.
+======2011-07-27======
+Yesterdays’ samples were sent for sequencing. Glycerol stock and plasmid prep of the O/N cultures from Yesterdays’. Mohammed and Pikkei made new LB medium. We also screened our yellow output (PcpcG2-B0034-amilGFP) and the band on the gel showed accurate length.
+Red output assembly
+PompC-tetR.inv + B0034-RFP in pSB1K3-B0032 and pSB1K3-B0034-RFP. We wanted to the test the output since the output can become either red or colorless.
+O/N -cultures of pSB2K3-I0500 (inverter test plasmid assembly).
+Evaluation
+The screening showed that the assembly of yellow output was done successfully.
+======2011-07-28======
+Today we did plasmid prep and glycerol stock of pSB2K3-I0500. Re-sreak of transformed and plated λ-Red plasmid and envZ amplicon (contains Kan-cassette)
+Screening of:
+YFI
+FixJ
+PfixK
+PcpcG2-B0034-BFP
+PcpcG2-B0034-YFP
+J23101-B0032-BFP
+J23101-B0032-YFP
+PompC-tetR.inv-B0034-BFP
+PompC-tetR.inv-B0034-YFP
+O/N cultures of pSB1A3-PcpcG2-B0034-BFP, pSB3K3-J23101-B0032-BFP
+pSB3K3-J23101-B0032-YFP and pSB1K3-PcpcG2-B0034-YFP.
+Evaluation
+The band on the gel for the screening showed correct length of all samples except for the PompC-tetR.inv-B0034-BFP.
+======2011-07-29======
+Johanna and Laura did glycerol stock and plasmid prep of the O/N - cultures from 2011-07-28. They also re-transformed the red output (PompC-tetR.inv + B0034-RFP) and did the experiment from 2011-07-27 again since we could come to a solid conclusion the first time.
+Blue output assembly
+PfixK+λC1.inv
+Blue sensor assembly
+B0034+FixJ
+B0034+YF1
+These assemblies were later transformed.
+O/N- cultures of pSB1K3-PcpcG2-B0034-YFP
+Evaluation
+There were no colonies on the re-streaked plate from yesterday. The result of the red output-assembly showed a plate with only white colonies (contain backbone pSB1K3-B0032) and one with both red and white (contain backbone pSB1K3-B0034-RFP). We picked the white colonies from the plate where the insert was put in pSB1K3-B0034-RFP- backbone which from now on be called red output with RFP (ROR). Hopefully this contains our insert and does not become induced by the TOP10-strain.
+======2011-07-30======
+Re-streak of all yesterday’s transformants. Today we also started making new competent TOP10 cells and inoculated them for the night. λ-Red plasmid is still processing slowly. But we ran a PCR to amplify envZ-KAN gene (Kan-cassette), followed by screening. O/N culture and screening of pSB1K3-PompC-tetR.inv-B0034-RFP. Point mutagenisis and O/N on pSB1K3-cph8_PstI since earlier mutagensis (first on) didn't work. Re-pleated, amplified and screening of earlier mutaded pSB1K3-Cph8_PstI.
+Evaluation
+The gel picture of amplified pSB1K3-cph8_PstI showed good result. It also seemed like the point mutagenesis of pSB1K3-cph8_PstI had an accurate length. The reason why we did a new mutagenesis is because we had a lot of trouble with our sensor before and we wanted to be on the safe side. The gel picture of envZ-KO and the red output didn't show any band and needs to be redone. At this stage the failure rate has increased leading reforms in the standard procedures so far. However, we are coming closer to the goal and need to keep to going.
+======2011-07-31======
+Testing the new top 10 competent cell by doing a transformation of PUC57 (with PfixK gene) and plating it on Amp plate. Running a gel of PfixK-λC1, B0034-YF1 and B0034-FixJ.
+(egentligen 1/8, men jag flytta för att det ska passa med summary)
+Colony PCR of B0034 - YF1 and B0034 – FixJ followed by screening. Additional screening of ROR and envZ-KO.
+PT5lac promotor arrived today
+Summary so far
+Blue output: B0034-amilCP (BOA) is assembled and sequenced and is ready for the final step. PfixK-λC1.inv is assembled but not yet sequenced. Blue output will soon be ready for the final assembly.
+Red output: Final assembly has been done several times with unsuccessful result. The reason for the problem is not clear. There seems to be a problem in the assembly of PompC-tetR.inv + B0034-RFP. Over and over again we can only see the band for tetR.inv on the gel. The sequence comes back with bad result every time. However, sequencing of the two individual components is good. Our screening method is might not be good enough.
+We decided to resume the attempt with mcherry and use it as a backup in case we would fail totally.
+Green output: Final assembly (pSB1K3-pcpcG2 + B0034-amilGFP) is done and the sequencing data showed perfect assembly. However, we are considering putting the final construct in a low copy plasmid (pSB3C5) because the promotor is slightly leaky.
+Chromophore assembly: Last assembly pSB4A5-PlacO-B0034-ho1 (CA2B) + B0034-pcyA-Term (CA2A) has been done but the sequencing result showed double peaks. Needs to be sequenced again. We will also check the previous assembly step CA2B by sending it for sequencing. At this stage we are considering an alternative assembly. We believe that ho1 activity decreases the concentration of Hem. Hence we will put the PLlacO promotor last.
+Red sensor: chp8 has been mutagenized two times to remove the illegal PstI site. The first time showed a large insert. Thus the mutagenezis has to be redone and send for sequencing.
+Green sensor: the mutation of ccaS has been a tough one. It has to be mutagenized two times and the first sequencing result showed an insert. Assuming that we were un-fortuned to pick a colony with inserts. We went back and choose other colonies. It was the right assumption because the second sequencing was correct. ccaR is already assembled with B0034.
+Blue sensor: Since the arrival of all the components the assembly of the blue sensor took of quit well. B0034-fixJ showed good sequencing results but B0034-YF1 showed no bands. Thus it needs to be assembled again!
+λ-red: Is lagging behind and has to be taken care of. All the attempts with envZ-KO so fare show modest or no results.
+Modelling: remains one of the toughest challenges. So far we haven’t found a model that can be easily implemented and understood. We are still working on it.
+Bioneer
+We sent an order to one of our sponsors Bioneer to synthesize tree color proteins that we can use as alternative output modules. aeBlue and cjBlue are different tones of blue and eforRed is a red protein.
+Test plasmid assembly
+Antionio and Thomas are focusing on making test plasmids. The aim to be able to meassure the output, promotor activity as well as inverter activity.
+== Week 7 ==
+======2011-08-01======
+Glycerol stock and plasmid preparation of PfixK-λC1.inv and red output. The chromophore (CA2A + CA2B) was PCR amplified together with B0034-RFP. Transformation of BBa_I732731.
+======2011-08-02======
+PCR purification of B0034-FixJ followed by plasmid preparation and freezing. Transformation of PT5lac. PCR purification of yesterday and preparation for sequencing. Re-streak of BBa_I732731 and screening of envZ-KO and B0034-YF1. O/N-culture of BBa_I732731.
+O/N-culture of pSB3C5, pSB4C5 and pSB1C5
+Green sensor assembly
+B0034 + ccaS  pSB1K3
+======2011-08-03======
+Plasmid preparation and glycerol stock of yesterday’s O/N-culture and cloning of pSB1K3-pcpcG2-B0034-amilGFP  pSB3C5. Re-streak of PT5lac and O/N culture of pSB4A5-CA3, pSB3K5, and PompC-tetR.inv.
+Output/promotor test plasmid assembly
+PompC-tetR.inv+B0034-BFP
+PompC-tetR.inv+B0034-YFP
+Blue output assembly
+PfixK+λC1.inv + B0034-amilCP
+Blue sensor assembly
+B0034 + YF1
+Sent for sequencing:
+CA2A, CA2B, PompC-tetR.inv, B0034-RFP, chp8_ PstI (new mut), B0034-FixJ, B0034-YF1.
+======2011-08-04======
+Transformation of chp8_ PstI, pcpcG2-B0034-amilGFP, plasmid preparation and glycerol stock of BBa_I732731, PompC-tetR.inv and CA3. Re-streak of blue sensor and output (B0034-YF1, PfixK- λC1-B0034-amilCP), PompC-tetR.inv-B0034-BFP and of PompC-tetR.inv-B0034-YFP. Screening of B0034-ccaS. Digestion of pSB1C3-B0034-YF1 with XbaI and with pstI- restriction enzyme. PCR amplification of CA3, screening and O/N-culture of PT5lac.
+Output/promotor test plasmid assembly
+PfixK- λC1.inv + B0034-YFP
+PfixK- λC1.inv + B0034-BFP
+Inverter test plasmid assembly
+Para/BAD + B0034-BFP
+Para/BAD + B0032-BFP
+Green sensor assembly
+B0034+ccaS  pSB1K3
+Red sensor assembly
+B0034+cph8  pSB1C3
+O/N- culture of PompC-tetR.inv, PompC-tetR.inv-B0034-RFP, B0034-ccaS
+Evaluation
+B0034-ccaS and PT5lac showed accurate length on the gel. Digestion of pSB1C3-B0034-YF1 because we wanted to check the length of the insert. Furthermore the sequencing result were good thus we can proceed with the following steps.
+======2011-08-05======
+Started with transformation of Para/BAD-B0034-BFP, Para/BAD-B0032-BFP, B0034-ccaS, B0034-cph8. We also did Plasmid prep and glycerol stock of PompC-tetR.inv-B0034-RFP, B0034-ccaS.
+We ran B0034-YF1, envZ-KO and CA3, on a gel and all the samples showed accurate length this time. PCR purification of B0034-ccaS.
+Screening of:
+pSB1A3-PompC-tetRinv-B0034-RFP
+pSB1A3-PompC-tetRinv-B0034-YFP
+Output/promotor test plasmid assembly
+PfixK- λC1.inv + B0034-YFP
+PfixK- λC1.inv + B0034-BFP
+Re-streak of chp8_ PstI. O/N- culture of B0034-YF1, PfixK- λC1-B0034-amilCP.
+Lambda red plasmid (Anto. pikkei…..)
+Evaluation
+No band shown after gel screening of pSB1A3-PompC-tetRinv-B0034-RFP and pSB1A3-PompC-tetRinv-B0034-YFP thus no need for O/N- Culture yet. The screening was redone with a lower annealing temperature.
+======2011-08-06======
+Re-streak of yesterday’s transformats. Successful screening of pSB1A3-PompC-tetRinv-B0034-BFP and pSB1A3-PompC-tetRinv-B0034-RFP. Hence, we proceeded with O/N-culture B0034-YF1 and PT5lac.
+Screening of;
+PfixK-λC1.inv-B0034-amilCP
+B0034-YF1
+PUC57-PT5lac
+Evaluation
+Only B0034-YF1 gave good result. New blue assembly of blue output is required.
+Green sensor assembly
+B0034-ccaS + B0034-ccaR  pSB4C5
+Followed by transformation
+======2011-08-07======
+Amplification of λ-red plasmid and PCR purification of envZ-KO and B0034-YF1. We also did plasmid preparation and glycerol stock of yesterday’s O/N-cultures and re-streak of B0034-ccaS-B0034-ccaR. We made new agar plates, and as usually plates with A, C and K resistance.
+Screening of:
+B0034-ccaS
+PfixK-λC1.inv-B0034-YFP
+PfixK-λC1.inv-B0034-BFP
+B0034-cph8
+PcpcG2-B0034-amilGFP
+PcpcG2-B0034-BFP
+J23101-B0032-BFP
+PcpcG2-B0034-YFP
+J23101-B0032-YFP
+Evaluation
+Good results for:
+PcpcG2-B0034-BFP
+J23101-B0032-BFP
+J23101-B0032-YFP
+PcpcG2-B0034-YFP
+PcpcG2-B0034-amilGFP
+B0034-ccaS
+Bad result for:
+PfixK-λC1.inv-B0034-YFP
+PfixK-λC1.inv-B0034-BFP
+B0034-cph8
+== Week 8 ==
+======2011-08-08======
+PCR purification of yesterday’s screens followed by glycerol and plasmid. Cloning of PT5lac  pSB1C3 and O/N-culture of Para/BAD-B0032-BFP and B0034-ccaS-B0034-ccaR.
+Output/promotor test plasmid assembly
+J23101 + B0034-BFP
+J23101 + B0034-YFP
+J23101 + B0034-amilCP
+J23101 + B0034-amilGFP
+======2011-08-09======
+Transformation of pSB3C5, pSB3K5 and pSB3S5 for later λ-red constructs. Additional transformation J231001-B0034-amilCP and J231001-B0034-amilGFP. The assembly of J23101 + B0034-BFP and J23101 + B0034-YFP didn’t succeed. Plasmid preparation of Para/BAD-B0032-BFP and B0034-ccaS-B0034-ccaR.
+Screening of:
+PfixK-λC1.inv-B0034-YFP, PfixK-λC1.inv-B0034-BFP and B0034-cph8 again. pSB1A3-PompC-tetRinv-B0034-YFP
+pSB1A3-PompC-tetRinv-B0034-RFP
+pSB1A3-PompC-tetRinv-B0034-BFP
+Para/BAD-B0032-BFP
+Para/BAD-B0034-BFP
+B0034-ccaS-B0034-ccaR
+Evaluation
+Good results only for:
+Para/BAD-B0032-BFP
+B0034-ccaS-B0034-ccaR
+New assemblies for the failed screenings
+Sent for sequencing:
+B0034-YF1- B0034-FixJ, PfixK-λc1-inv-B0034-amilCP, PT5lac, PfixK-λc1 inv-B0034-YFP, B0034-ho1-B0034-PcyA-term, RBS-cph8, B0014-PlacO, Para/Bad-RBS-BFP, pSB3K3-K098010 and pSB1A3-PompC-tetRinv-B0034-RFP
+======2011-08-10======
+Re-streak of J231001-B0034-amilCP and J231001-B0034-amilGFP. New assemblies made for failed sequencing followed by transformation.
+Blue output assembly
+New assembly of PfixK-λC1.inv + B0034-amilCP
+Followed by transformation
+Output/promotor test plasmid assembly
+PfixK-λC1.inv + B0034-BFP
+PfixK-λC1.inv + B0034-YFP
+Followed by transformation
+======2011-08-11======
+Evaluation
+The sequence data for Para/BAD+B0032-BFP was hard to interpret. The sequence data of the new assembly of pSB4K5-PLlacO-B0034-ho1, pSB1K3-PompC-tetRinv-B0034-RFP and the old assembly of pSB1K3-PompC-tetRinv-B0034-RFP didn’t show satisfying result. However, B0034-RFP segment is clear. The rest of the samples that we sent for sequencing showed good result.
+Re-streak of yesterday’s transformants and pSB3C5-FRT backbone and pSB3S5-FRT.
+O/N-cultures of
+pSB1AK3-B0014
+pSB1A2-R0011
+pSB1K3-J23101-B0034-amilCP
+pSB1K3-J23101-B0034-amilGFP
+Red sensor assembly
+New assembly of B0034+cph8  pSB1C3
+Followed by transformation
+Making Lambda Red compatible BioBrick plasmids
+Repeat cloning of SpecR and KanR-amplicon
+======2011-08-12======
+Re-streak of yesterday’s transformants and of A, C, K-backbone.
+Screening of:
+PT5lac
+PfixK-λC1.inv-B0034-amilCP
+PfixK-λC1.inv-B0034-YFP
+PfixK-λC1.inv + B0034-BFP
+Plasmid prep and frozen stock of:
+PT5lac
+PfixK-λC1.inv-B0034-amilCP
+PfixK-λC1.inv-B0034-YFP
+pompC-tetR.inv-B0034-RFP
+J23101-B0034-amilGFP
+J23101-B0034-amilCP
+Assembly of our coupled solution for chromphore and sensor
+B0014+PLlacO  pSB1C3
+Output/promotor test plasmid assembly
+New assembly of PfixK-λC1.inv+B0034-BFP
+Blue sensor assembly
+RBS-YF1+RBS-fixJ
+Followed by transformation
+Chromophore assembly
+RBS-ho1+RBS-pcyA-B1001 (term)
+Followed by transformation
+Evaluation
+Since the band of PfixK-λC1.inv + B0034-BFP showed wrong length we need to do the assembly of again. The rest of the samples showed the right length.
+Making Lambda Red compatible BioBrick plasmids
+Ligation of Spec and KanR……..
+Transformation of:
+pSB1A10, the plasmid that are going to be used for the inverter test plasmid.
+Transformation of different inverter components taken from the BioBrick-kit.
+Followed by transformation of Lambda red components (SpecR, KanFRT).
+======2011-08-13======
+Re-streak of pSB1A10 and RBS-YF1+RBS-fixJ. Additional re-streak of pSB3C5-FRT because of the first attempt failed.
+Output/promotor test plasmid assembly
+J23101 + B0034-BFP (new assembly)
+J23101 + B0034-YFP (new assembly)
+PT5lac + B0032-YFP  pSB3K3
+PT5lac + B0032-BFP  pSB3K3
+PT5lac + B0034-BFP  pSB1K3
+PT5lac + B0034-YFP  pSB1A3
+Red sensor assembly
+New assembly of B0034+chph8
+Inverter test plasmid assembly
+New assembly of PARA/BAD+B0032
+PARA/BAD+B0034
+Followed by transformation
+Amplification of pfixK-λC1.inv-B0034-amilCP with touch down PCR, since older gel picture showed two bands.
+Transformation of:
+PfixK-λC1.inv-B0034-BFP
+pSB1C3-B0014-PLlacO
+pSB3K3-K098010 (Harvard's chromo mod)
+O/N-culture of:
+pSB1A3-backbone
+pSB1C3-backbone
+pSB1K3-backbone
+Evaluation
+Still, even though using a touch down approach, we saw one weak second band for the pfixK-λC1.inv-B0034- amilCP amplicon. Next time we will change the protocol with a slightly higher starting and ending temperature.
+======2011-08-14======
+O/N culture of pSB3C5-FRT (λ-red plasmid) and screening of RBS-YF1+RBS-fixJ.
+Re-streak of
+PfixK-λC1.inv-B0034-BFP
+B0014-PLlacO
+B0034+chph8
+Antonio
+Digest; pSB3C5SpecR
+pSB3X5Kan-FRT
+Plasmid prep and glycerol stock of
+pSB1A3-backbone
+pSB1C3-backbone
+pSB1K3-backbone
+Second amplification attempt of PfixK-λC1.inv-B0034-BFP with an alter touch down PCR protocol. Re-streak of yesterday’s transformants.
+Transformation of
+pSB1A2-J06504 (mcherry, from kit)
+pSB1K3-J23101-B0034-YFP
+pSB1K3-pT5lac-B0034-BFP
+pSB1K3-pT5lac-B0034-YFP
+pSB3K3-pT5lac-B0032-YFP
+pSB3K3-pT5lac-B0032-BFP
+== Week 9 ==
+======2011-08-15======
+Plasmid prep and glycerol stock of pSB3C5-FRT, RBS-YF1+RBS-fixJ and pSB1A10. Re-streak of yesterday’s transformations.
+Screening and O/N of:
+B0034+chph8
+pSB3K3-K098010
+B0014-PLlacO
+PT5lac-B0034-YFP
+RBS-ho1-RBS-pcyA-B1001
+Output/promotor test plasmid assembly                                                                                                  pSB1A3-PompC-tetRinv + B0034-RFP
+Followed by transformation
+Tomas Transformation and re-streak
+pSB3C5SpecR
+pSB3X5Kan-FRT
+Evaluation
+Satisfying screening results. However, pSB3C5-FRT, RBS-YF1+RBS-fixJ failed again. Wrong band on the gel.
+======2011-08-16======
+PCR purification of yesterday’s screens
+Antonio
+Digest; pSB3C5-Kan-FRT with Sal1 & Sac1
+Ligation of al ready digested components:
+Output/promotor test plasmid assembly
+pSB1K3-PT5lac + B0032-YFP
+pSB1K3-PT5lac + B0034-BFP
+pSB1K3-PT5lac + B0034-BFP
+======2011-08-17======
+Transformation of:
+PomPC-tet.R.inv-B0034-amilCP
+PT5lac-B0034-BFP
+PT5lac-B0032-YFP
+PT5lac-B0032-BFP
+KanR-FRT
+SpecR
+O/N-cultures of:
+PfixK-λ C1-B0034-BFP
+B0014
+mCherry
+PT5lac-B0034-YFP
+J23101-B0034-YFP
+J23101-B0034-BFP
+Red output assembly
+PompC-tetRinv + B0034-amilCP --> pSB1A3
+A test to see if our assembly method to put our red output together should be done in a diffrent way.
+Red sensor assembly
+B0034-Cph8 + B0014 --> pSB3T5
+Digestion of B0034-Cph8+B0014-PlacO --> pSB3T5
+The promotor that we have been using might be toxic to the cell, therefor we are trying out different approaches.
+Colony PCR of pSB1A3-PfixK-λ C1-B0034-BFP
+======2011-08-18======
+Glycerol stock of:
+PfixK-λC1.inv-RBS-BFP
+mCherry
+PT5lac-RBS-YFP
+J23101-RBS-BFP
+J23101-RBS-YFP
+Restreak of:
+PompC-tetR.inv-RBS-amilCP
+SpecR
+KanR-FRT
+PT5lac-B0032-YFP
+PT5lac-B0034-BFP
+PT5lac-B0032-BFP
+Transformation of:
+RBS-Cph8-B0014-PlacO
+Ligation of pSB3T5-B0034-Cph8+B0014-PlacO over night.
+Red output assembly
+Assembly of:
+RBS+mCherry --> pSB3C5 (Alternative for our red output.)
+RBS-cph8+B0014 --> pSB3T5
+======2011-08-19======
+Re-streak of pSB3T5-RBS-cph8-B0014-PlacO
+O/N-culture of J23113 and pSB4K5-LacIq (Repressor)
+Screening of:
+PfixK-λC1.inv-RBS-BFP
+PT5lac-B0032-YFP
+PT5lac-B0032-BFP
+Transformation of :
+pSB1A3-PompC-tetRinv-B0034-BFP
+pSB1K3-PompC-tetRinv-B0034-YFP
+pSB3K5 FRT backbone (for lambda -red)
+pSB3T5-RBS-cph8-B0014-PLlacO
+======2011-08-20======
+Re-streak of:
+PT5lac-RBS-YF1-RBS-FixJ
+RBS-cph8-B0014-PlacO
+PompC-tetR.inv-B0034-BFP
+PompC-tetR.inv-B0034-YFP
+RBS-cph8-B0014
+Plasmid prep and glycerol stock of:
+PT5lac-B0032-YFP
+PT5lac-B0032-BFP
+laqIq
+J23113
+Red output assembly
+New assembly of
+B0034+mCherry --> pSB1C3
+since earlier transformation didn't show any colonies.
+O/N-cultures of:
+KanR-FRT
+SpecR
+B0034-cph8-B0014-PLacO
+PompC-tetR.inv-B0034-BFP
+Screening of:
+SpecR
+KanR-FRT
+pSB3C5-FRT
+RBS-cph8-B0014-PLacO
+PompC-tetR.inv-B0034-BFP
+PT5lac-B0034-BFP
+Evaluation
+The screening showed accurate lenght of the following samples:
+KanR-FRT
+SpecR
+B0034-cph8-B0014-PLacO
+PompC-tetR.inv-B0034-BFP
+======2011-08-21======
+Transformation of B0034-mCherry
+Frozen stock and plasmid prep of:
+B0034-cph8-B0014-PlacO
+SpecR
+KanR-FRT
+== Week 10 ==
+======2011-08-22======
+Another, new transformation of B0034-mCherry, since yesterdays transformation didn't show anything on the plate.
+Plating of EnvZ-KO (clone one and two)
+Screening of:
+PompC-tetR.inv-B0034-BFP
+PompC-tetR.inv-B0034-YFP
+RBS-cph8-B0014
+Evaluation
+Screening of samples,see above, showed accurate band length for all assemblies except for PompC-tetR.inv-B0034-BFP.
+======2011-08-23======
+Samples prepared and sent for sequencing:
+PompC-tetR.inv-B0034-YFP
+RBS-cph8-B0014-PlacO
+RBS-cph8-B0014
+Blue sensor assembly
+J23101+RBS-YF1-RBS-FixJ
+Green output assembly
+J23101+B0034-ccaS-B0034-ccaR --> pSB3K3
+Red sensor assembly
+J23101+B0034-cph8 --> pSB3K3
+Re-digestion and ligation of:
+PfixK-λ C1--> pSB1C3
+B0034-BFP --> pSB1K3
+Re-ligation of B0034-mCherry --> pSB1C3
+======2011-08-24======
+Plasmid prep and glycerol stock of:
+B0034-CcaS-B0034-CcaR
+J23113
+Transformation of:
+pSB3K3-J23101-RBS-cph8
+psB3C5-J23101-RBS-YF1-RBS-fixJ
+RBS-mCherry
+PfixK-λ C1-B0034-BFP
+J23101-RBS-cph8
+J23101-RBS-YF1-RBS-FixJ
+PT5lac-RBS-YF1-RBS-FixJ
+pSB1K3-J23101-B0034-CcaS-B0034-CcaR
+The Chromopher
+Assembly of J23113-RBS-ho1-RBS-pcyA-B1001, followed by transformation.
+Screening of:
+B0034-CcaS-B0034-CcaR,
+RBS-YF1-RBS-fixJ, used to compare the band length with, PT5lac-RBS-YF1-RBS-FixJ
+Evaluation
+Screening showed right band-length of PT5lac-RBS-YF1-RBS-FixJ and of B0034-CcaS-B0034-CcaR.
+======2011-08-25======
+Screening of :
+J23101-B0032-BFP
+J23101-B0032-YFP
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+FRT-lacIq
+EnvZ-KO
+O/n- culture of:
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+mCherry
+PT5lac-RBS-YF1-RBS-FixJ
+Re-streak of:
+J23101-RBS-YF1-RBS-FixJ
+J23101-RBS-cph8
+J23101-B0034-ccaS-B0034-ccaR
+J23113-RBS-ho1-RBS-pcyA
+PfixK-λ C1-B0034-BFP
+Evaluation
+Gel picture of screened samples showed good length except for EnvZ-KO.
+======2011-08-26======
+Red output assembly
+Redo assembly of B0034+mCherry --> pSB1C3
+Screening of:
+J23101-RBS-YF1-RBS.FixJ
+PfixK-λ C1-B0034-BFP
+J23101-RBS-cph8
+FRT-lacIq
+O/n-culture of:
+J23101-RBS-YF1-RBS-FixJ
+PfixK-λ C1-B0034-BFP
+J23101-RBS-cph8
+FRT-lacIq
+Evaluation
+Very bad gel picture due to the agarose.
+======2011-08-27======
+Screening and O/n-culture of:
+J23101-B0034-ccaS-B0034-ccaR
+J23113-RBS-ho1-RBS-pcyA-B1001
+envZ-KO
+Transformation of:
+B0034-mCherry
+Pfixk
+======2011-08-28======
+Output/promotor test plasmid assembly
+PcpcG2+B0032-BFP --> pSB3K3
+PcpcG2+B0032-YFP --> pSB3K3
+PlacO+B0032-BFP --> pSB3K3
+PlacO+B0032-YFP --> pSB3K3
+J23113+B0032-BFP --> pSB3K3
+J23113+B0032-YFP --> pSB3K3
+Re-streak of:
+J23101-B0034-amilCP
+B0034-ccaS-B0034-ccaR-RBS-cph8-B0014
+RBS-ccaS-RBS-ccaR-RBS-cph8-B0034-PlacO
+PfixK
+== Week 11 ==
+======2011-08-29======
+Screening of:
+pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014
+pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014-PLlacO
+PfixK
+O/n-culture of:
+J23113-RBS-ho1-RBS-pcyA-B1001
+J23101-RBS-cph8
+FRT-lacIq
+J23101-B0034-amilCP
+B0034-ccaS-B0034-ccaR-B0034-cph8-B0014
+B0034-ccaS-B0034-ccaR-B0034-cph8-B0014-PlacO
+PfixK
+Blue backbone assembly
+J23101-B0034-amilCP+B0034 --> pSB1C3
+J23101-B0034-amilCP --> pSB1C3
+Red output assembly
+B0034+mcherry --> pSB1C3
+Transformation of:
+pSB3K3-PcpcG2-B0032-BFP
+pSB3K3-PcpcG2-B0032-YFP
+pSB3K3-PLlacO-B0032-BFP
+pSB3K3-PLlacO-B0032-YFP
+pSB3K3-J23113-B0032-BFP
+pSB3K3-J23113-B0032-YFP
+Evaluation
+Couldn't see any band on the gel for PfixK and the bands for samples pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014 and
+pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014-PLlacO had the wrong length.
+======2011-08-30======
+Another screening of
+pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014
+pSB3K3-B0034-ccaS-B034-ccaR-B0034-cph8-B0014-PLlacO
+but this time using both taq and phusion polymerase.
+Transformation of
+J23101-B0034-amilCP-B0014
+B0034-mcherry
+J23101-B0034-amilCP
+PCR purification of:
+RBS-cph8
+J23113-RBS-ho1-RBS-pcyA-B1001
+Re-streak of:
+pSB3K3-PcpcG2-B0032-BFP
+pSB3K3-PcpcG2-B0032-YFP
+pSB3K3-PLlacO-B0032-BFP
+pSB3K3-PLlacO-B0032-YFP
+pSB3K3-J23113-B0032-BFP
+pSB3K3-J23113-B0032-YFP
+Output/promotor test plasmid assembly
+PfixK+B0032-BFP --> pSB3K3
+PfixK+B0032-YFP --> pSB3K3
+======2011-08-31======
+Screening of:
+pSB3K3-PcpcG2-B0032-BFP
+pSB3K3-PcpcG2-B0032-YFP
+pSB3K3-PLlacO-B0032-BFP
+pSB3K3-PLlacO-B0032-YFP
+pSB3K3-J23113-B0032-BFP
+pSB3K3-J23113-B0032-YFP
+======2011-09-01======
+Transformation of:
+PfixK-B0032-BFP
+PfixK+B0032-YFP
+PCR amplification of FRT-lacIq
+======2011-09-02======
+Re-streak of:
+PfixK-B0032-BFP
+PfixK-B0032-YFP
+======2011-09-04======
+O/n- culture of:
+J23101-B0032-BFP
+J23101-B0032-YFP
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+PcpcG2-B0032-BFP
+PcpcG2-B0032-YFP
+PLlacO-B0032-BFP
+PLlacO-B0032-YFP
+J23113-B0032-BFP
+J23113-B0032-YFP
+== Week 12 ==
+======2011-09-05======
+Screening of:
+FRT-lacIq
+Pt5lac-RBS-YF1-RBS-FixJ
+J23101-RBS-YF1-RBS-FixJ
+Evaluation
+The screened samples showed right length for FRT-lacIq but not for the rest.
+======2011-09-07======
+Plasmid prep and frozen stock of:
+J23101-B0034-amilCP
+J23113-RBS-ho1-RBS-pcyA-B1001
+pGEM-cjBlue
+pGEM-eforRed
+J23101-RBS-amilCP-B0014
+Transformation of
+pcpcG2
+PompC-tetRinv
+B0034-CcaS
+CA2A
+B0032-BFP
+amilGFP
+B0034-ccaR
+YF1
+FixJ
+RBS-ho1-RBS-pcyA-B1001
+RBS-YF1-RBS-fixJ
+B0034-CcaS-B0034-CcaR
+======2011-09-08======
+Assembly of:
+B0034 + eforRed
+B0034 + pompC-tetR
+B0034 + mcherry
+J23101-B0034 + eforRed
+J23101-B0034 + cjBlue
+Re-streak of yesterday’s transformants.
+PCR amplification of:
+B0034-cph8-B0014-PLlacO
+J23101-RBS-YF1-RBS-fixJ
+======2011-09-09======
+O/n-culture of
+J23101-RBS-amilCP-B0014
+PCR amplification:
+pcpcG2
+CA2A
+B0032-BFP
+B0034-BFP
+YF1
+FixJ
+amilGFP
+cph8_PstI
+B0034-CcaS
+RBS-ho1-RBS-pcyA-B1001
+Re-streak of:
+PompC-tetR-B0034
+B0034-ccaR
+B0034-YF1-B0034-fixJ
+J23101-B0034-eforRed
+J23113-RBS-ho1-RBS-pcyA-B1001
+======2011-09-10======
+O/N-cultur of pSB1C3-J23101-RBS-amilCP-B0014. Digest of J23101-RBS-YF1-RBS-fixJ, pSB4A5-PT5lac-RBS-YF1-RBS-FixJ.Screening of yesderday’s amplicons
+Colony PCR of:
+J23101-B0034-eforRed
+J23113-RBS-ho1-RBS-pcyA-B1001
+B0034-YF1-B0034-fixJ
+B0034-ccaR
+PompC-tetRinv-B0034
+PT5lac-RBS-YF1-RBS-FixJ
+J23101-RBS-YF1-RBS-fixJ
+Evaluation
+The gel showed no bands. Suspect that something might be wrong with the gel. Thus, we did a new batch.
+======2011-09-11======
+New screening attempt of yesterday’s PCR. It was difficult to see anything significant from the other bands except from that of the digested blue sensor. PT5lac-B0034-YF1-RBS-FixJ and J23101-B0034-YF1-RBS-fixJ.
+Tranformation of PT5lac-B0034-YF1-B0034-FixJ, pSB1K3-PfixK-λ C1-B0034-amilCP and pSB1K3-PfixK-λ C1-B0034-YFP in MG1655 strain.
+PCR amplification of PT5lac-B0034-YF1-B0034-FixJ for sequencing.
+== Week 13 ==
+======2011-09-12======
+Cloning of constructs into pSB1C3 to be sent to the registry:
+amilCP
+PcpcG2-B0034-BFP
+J23101-B0032-BFP
+J23101-B0032-BFP
+J23101-B0032-YFP
+PcpcG2-B0034-amilGFP
+B0034-CcaS
+PcpcG2-B0034-YFP
+PfixK-λ C1-B0034-amilCP
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+-09-12
+Re-streak of:
+B0034-cjGreen
+B0034-eforRed
+PfixK-λ C1-B0034
+Prepare agar stab BL-21 E.coli strains to be sent to Bilkent Unam Unversity (Turkey, Ankara)
+======2011-09-13======
+Transformation of the parts to be sent to the registry. Re-streak of pSB1K3-J23101-B0034-cjGreen followed by O/N.
+PCR and gel of;
+J23101-B0034-CcaS-B0034-CcaR
+PfixK-B0032-BFP
+PfixK-B0032-YFP
+PT5lac-RBS-YF1-RBS-FixJ
+J23101-RBS-YF1-RBS-fixJ
+Screening showed good results.
+======2011-09-14======
+Re-streak of:
+pSB1K3-PfixK-λ C1-B0034-YFP
+pSB1K3-PfixK-λ C1-B0034-amilCP
+pSB1K3-J23101-B0034-CcaS-B0034-CcaR
+pSB1C3-amilCP
+pSB1K3-PfixK-λ C1-B0034
+pSB1C3-PcpcG2-B0034-BFP
+pSB1C3-PcpcG2-B0034-YFP
+pSB1C3-PcpcG2-B0034-amilGFP
+pSB1C3-B0034-CcaS
+pSB1C3-PfixK-λ C1-B0034-amilCP
+pSB1C3-PT5lac-B0032-BFP
+pSB1C3-PT5lac-B0032-YFP
+pSB1C3-J23101-B0032-BFP
+pSB1C3-J23101-B0032-YFP
+Assembly of:
+J23101-B0034 + pGEM-eforRed  pSB1K3
+Cloning of:
+pGEM-eforRed  pSB1C3
+pGEM-cjGreen  pSB1C3
+======2011-09-15======
+O/N-culture and glycerol stock of yesterday’s re-streaks.
+Plasmid prep of:
+RBS-YF1-RBS-fixJ
+amilGFP
+B0034-ccaR
+B0032-BFP
+B0032-YFP
+J23113-B0032-BFP
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+PcpcG2-B0032-BFP
+PcpcG2-B0032-YFP
+PLlacO-B0032-BFP
+PLlacO-B0032-YFP
+J23101-B0032-BFP
+Assembly of:
+J23113 + B0032-YFP
+J23101 + B0034-YFP
+======2011-09-16======
+Re-streak of:
+cjGreen
+pSB1C3-eforRed
+J23101-B0034-cjGreen
+J23101-B0034-eforRed
+J23113-B0032-YFP
+J23101-B0034-YFP (YFP from part registry)
+Screening of:
+pcpcG2
+B0034-BFP
+YF1
+FixJ
+cph8_PstI
+B0034-CcaS
+PfixK-λ C1-B0034
+J23101-B0032-YFP
+PcpcG2-B0034-YFP
+PcpcG2-B0034-amilGFP
+B0034-CcaS
+PfixK-λ C1-B0034-amilCP
+cph8_PstI
+PfixK-λ C1-B0034
+pSB1C3-amilCP
+PcpcG2-B0034-BFP
+J23101-B0032-BFP
+J23101-B0032-YFP
+PcpcG2-B0034-YFP
+PcpcG2-B0034-amilGFP
+B0034-CcaS
+PfixK-λ C1-B0034-amilCP
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+amilCP
+plasmid prep and O/n-culture of:
+amilCP
+PcpcG2-B0034-BFP
+J23101-B0032-BFP
+PfixK-λ C1-B0034-amilCP
+PT5lac-B0032-BFP
+PT5lac-B0032-YFP
+Evaluation
+Everything that we had screened showed the right length except for YF1 and cph8_PstI.
+======2011-09-17======
+Re-streak of:
+J23101-B0034-YFP (Erik Gullbergs YFP)
+YFP (Erik Gullbergs YFP)
+Blue sensor assembly
+J23101+RBS-YF1-RBS-fixJ   pSB4C5
+Output/promotor test plasmid assembly
+J23101+B0034-YFP  pSB3K3 (Erik Gullbergs YFP)
+O/n-culture of:
+Pt5lac-RBS-YF1-RBS-fixJ
+RFP
+amilGFP
+amilCP
+cjGreen
+J23101-B0034-cjGreen
+J23113-B0032-YFP
+{{Template:Uppsala-SwedenTemplatefooter}}