Reporters and Promoters

Generating standard constructs for ease of biobrick characterisation

and submission

03/08: Due to lack of success with E1010 BioBrick, gel electrophoresis was carried out to confirm presence of DNA in the samples. Only OmpR and RBS+RFP, from yesterday’s transformations, yielded positive results. Overnights (o/n) were made of these transformants. Digests were made of the following BioBricks overnight:

Strong Promoter (S.promoter) (J23119)Weak Promoter (W.promoter) (J23106)Strong Ribosome Binding Site (S.RBS) (B0030)Bluf (K238013)OmpF (K116500)LOV2 (PCR)LATH (PCR)PDE (PCR)RANA (PCR)Lac Z + S.RBS 04/08: Ligations of... S.Promoter + Strong RBSBluf Promoter + Strong RBSOmpF + Strong RBSW.Promoter + Strong RBS

- Don’t have Weak RBS or Medium promoter

4 tubes for 4 hour ligation 4 hours4 tubes for 10 min ligation were actually left for 25 minutesAlso set up ligations in the afternoon

Ligations set up in the afternoon were:

S.Promoter - RBS-RFP

W.Promoter - RBS-RFPBluf Promoter - RBS-RFPOmpF Promoter - RBS-RFP

Transformations of 12 ligations into 37⁰C incubator. 05/08: Growth on all ligation plates from yesterdayMiniprep of o/n’s from yesterday:

E1010 x 3ILOV + RSN2 x 2

PUC x 1Strong RBS + YFP + TTWeak RBS + YFP + TTWeak RBS x 3

There were no sterile eppendorfs so used straight from the packet to elute the DNA into. 08/08: Plated S.Promoter from glycerol stocks. Put into 37ºC incubator.

09/08: Overnights of Strong Promoter (x3) and Strong RBS (x3) were made and put into 37⁰C shaker incubator.

Digested:

S.Promoter S and P Bluf Promoter S and P OmpC Promoter S and P pBAD Promoter S and P Strong RBS X and P Weak RBS X and P(Used 1μl of PstI and 0.75 μl of XbaI)

Ligated according to Louise’s Protocol. Put into -20ºC freezer overnight. 10/08: Transformation of the 8 ligations from the 9th.Glycerols and minipreps of the following were made and stored n the -80ºC: w.prom +RBS + RFP, S.Prom + RBS +RFP, Bluf+RBS+RFP, ompf+RBS+RFP, W.Promoter+S.RBS, S.Promoter+S.RBS, Bluf+S.RBS, Bluf+S.RBS, ompf+S.RBS. These minipreps were also digested and ran on a gel. 11/08: Digest of RFP at SpeI and PstI was made, whilst a digest of the double terminator was also made but at XbaI and PstI sites. These were done following standard protocol in an attempt to ligate them into a construct which was plated o/n. Transformations from 10th were set up o/n. 12/08: The o/n’s of the 8 ligations from the 10th were put through a miniprep and stored. Also made glycerol stocks of these, but they are labelled wrong (BL21 instead of Top10). Ligation attempt number 1: insert (wProm, sProm, Bluf, OmpF) and vector (RBS+RFP) were ligated following the standard protocol BUT weren’t extracted from a gel. visualizing the ligations on a the gel resulted in a ‘gel of ladders’. failed attempt. 15/08: Restriction digests of the following:S.promoter (AlwnI+SpeI)OmpC （AlwnI+SpeI)Bluf (AlwnI+SpeI)pBAD (AlwnI+SpeI)Strong RBS(AlwnI+XbaI)Weak RBS (AlwnI+XbaI)DNase (AlwnI+SpeI)TT (AlwnI+XbaI)

Subsequent running of a gel, as well as gel extraction and ligations were carried out; each promoter type was ligated to each RBS once apart from the Strong Promoter, which did not show up on the gel. A similar incident happened with the prospective DNase-TT construct: TT did not appear and so this construct was postponed to a later date.

Transformations were then created and plated out later that night.

Digest of: RBS+RFP with EcoRI and SpeI Strong Terminator with EcoRI and XbaI

when ran on a gel the fragment pattern didn’t show up as expected.digest was repeated with different minipreps.

Again the size of the fragments and the pattern of the bands didn’t look as expected.the concentration of strong Terminator was too low to be properly visualised on the gel.the experiment was repeated with 1ul of R.E. and 5ul of Terminator DNA.

the 3rd gel, again, showed a weird fragment pattern. through the analysis of the controls on the gel we realised the RBS+RFP biobrick was lacking the standardised suffix and that any attempt to restrict t with SpeI or PstI would have failed. 16/08: Of the plated transformations, only the control and the OmpC plates produced colonies. We believed that the BL21 cells were to blame! Also the EnvZ negative cells from the BioBrick registry arrived and plated on kanamycin plates. Growth was positive.

Re-attempted the transformations from the 15th, this time using Top 10 cells instead of BL21 cells. Also added a new prospective construct to the mix: RFP-TT. We digested OmpF, sRBS-YFP-TT and wRBS-YFP-TT with SpeI-PstI, XbaI-PstI and XbaI-PstI respectively.Samples and controls were run on a gel and the appropriate fragments were extracted following the kit protocol and then ligated. The resulting two ligations (promoter-weak/strong reporter) were stored at -20ºC. 17/08: Growth on all ligation plates that were re-transformed using Top10, except for: S. Promoter+ S.RBSS. Promoter+ W.RBSDNase+TT

However this was expected as the S.Promoter and the DNase didn’t appear on the gel from 15th August.

And so begins a new digest in an attempt to save the Strong promoter and DNase, as well as bring in Weak Promoter for good measure:

S.Promoter (1)S.Promoter (2)W.Promoter (1)W.Promoter (2)S.RBS (1)W.RBS (1)DNase (1) & (3)TT

Yet when the gel was run, S.promoter, TT and S.RBS were all absent. Gel extraction still went

ahead [with just W.Promoter (1), W.Promoter (2), W.RBS, DNase (1) and DNase (3)] and ligations were made.

Also set up overnights of “re-transformations” from the 16th. digestion of: pBAD with EcoRI-SpeI strong Promoter with EcoRI-SpeI weak Promoter with EcoRI-SpeI strong RBS-YFP-TT (sYFP) with PstI-SpeI weak RBS-YFP-TT (wYFP) with PstI-SpeI pSB1C3 with EcoRI-PstI

1ul of each of the resulting digests was used in each of the following reactions (no gel extraction was carried out since this ligation was done following the BioBrick assembly protocol):- pSB1C3 + pBAD + sYFP- pSB1C3 + pBAD + wYFP- pSB1C3 + weak Promoter + sYFP- pSB1C3 + weak Promoter + wYFP- pSB1C3 + strong Promoter + sYFP- pSB1C3 + strong Promoter + wYFP

these ligations were carried out as described, transformed in Top10 E.Coli and plated on Chloramphenicol-containing agar plates.

the ligated constructs left in the -20 overnight were transformed into Top10.RFP+TT, OmpF+sYFP and OmpF+wYFP were plated on Carbenicillin plates.

biobricks extracted from minipreps ( strong terminator B1006, Strong RBS B0030 and strong Promoter J23119) were transformed as well into Top10 and plated on Carbenicillin. 18/08: Miniprepped the o/n’s and made glycerols of them. It was decided that the chase for the nonexistent S.Promoter was only hindering our progress, and so a brand new one was taken from the original wells (J23118- S.Promoter #2) and transformed using Top10. Ligations of W.Prom+W.RBS were also created using Top10, and both were plated o/n. The biobrick assembly ligation didn’t produce any colony on the plates therfore the same reactions were carried out by digesting the promoters with SpeI-PstI and the YFP constructs with XbaI-PstI. the digests were then run on a gel and extracted.

The strong Promoter miniprep didn’t contain enough DNA to be visualised on the gel, not even after incubation of the gel in Ethidium Bromide.

Leftover digests from yesterday were run on the same gel to check whether the submission plasmid pSB1C3 was actually linearized or not. the band for this plasmid was very faint but about the right size (2070bp); this experiment is going to be repeated.

The excised and gel purified fragments were ligated following Louise’s protocol for ligation.The obtained samples were then stored in the -20, ready to be transformed the next day.

Ligations: - pBAD + sYFP

- pBAD + wYFP - strong Promoter + sYFP - strong Promoter + wYFP

Overnights were done of all of the successful transformations obtained yesterday. 19/08: An investigative gel was run for the S.prom, DNase, S.RBS, Double Terminator and RFP. Each BioBrick was run as Uncut, Single cut (AlwnI), Single cut (SpeI or XbaI) and Double cut. Results were mixed; both DNase and RFP returned positive results but showed multiple bands in all of their lanes, whilst the Strong Promoter, Strong RBS and the Double Terminator returned negative results. 22/08: Ligated several promoter constructs to an RFP + TT construct. The promoter constructs used were: OmpC+S.RBSOmpC+W.RBS pBAD+S.RBSpBAD+W.RBS BLUF+S.RBSBLUF+W.RBS W.promoter+S.RBSW.promoter+W.RBS S.promoter+S.RBSS.promoter+W.RBS Transformations of these were made according to standard protocol. More transformations of Top10 chem. comp. cells with:- OmpC-wYFP- OmpC-sYFP- sProm-wYFP- sProm-sYFP- wProm-wYFP- wProm-sYFP- pBAD-wYFP- pBAD-sYFP

Digestion of Bluf (SpeI-PstI) and sYFP, wYFP (XbaI-PstI).the digests were run on a gel and the relevant bands were extracted.

Digestion of OmpC, OmpF, sProm, wProm and pBAD (each ligated to strong and weak YFPs)with EcoRI-PstI to check for success of these ligations.

the gel failed to visualise any band except the ladder. 23/08: Transformations from the 22nd failed; few colonies were recorded and none were red. Around 18 hours of growth are required for visible presence of RFP-producing colonies. Minipreps of the following were made:W.promoter (1) + W.RBS [x3]W.promoter (2) + W.RBS [x2]J23118 [x6] From the transformation plates, constructs were visualized to check for RFP fluorescence. These constructs were: OmpC+RBS+RFP+TTS.promoter+RBS+RFP+TTpUC control Results were inconclusive... Set up o/n’s of the successful plates from the 22nd: W.promoter+W.RBS [x3 red colonies]W.promoter+W.RBS [x1 white colony]W.promoter+S.RBS [x1]S.promoter+S.RBS [x3] all the transformation from yesterday containing s/wYFP worked.overnights were made in triplicates for each plate.

Transformation of Bluf-sYFP and Bluf-wYFP in top 10 (plated on carbenicillin agar).Transformation of : BBa_K274120 (lycopene pathway) BBa_K274220 (beta-Carotene pathway)t’ns plated on kanamycin agar.

24/08: Made minipreps of the folowing: S.promoter+S.RBS+RFP+TT [x3]OmpC+W.RBS+RFP+TT [x3 RED]OmpC+S.RBS+RFP+TT [x1] We digested the following with EcoRI and PstI: S.promoter #1S.promoter #2S.promoter #3

OmpC+W.RBS #1OmpC+W.RBS #2OmpC+W.RBS #3OmpC+W.RBS#4OmpC+S.RBS #1 pBAD+S.RBS #1pBAD+W.RBS Bluf+S.RBSBluf+W.RBS W.promoter+S.RBSW.promoter+W.RBSS.promoter+W.RBS These digests were then cut and visualised under the transilluminator, in a bid to determine whether RFP had been inserted. RESULTS: No bands were observed at the desired lengths. The following were also digested and run on a gel, in the hope to gel extract them and carry out ligations: OmpF #1OmpF #2OmpF #3S.promoterW.promoterRFPTTS.RBSW.RBS However, there were no visible bands for TT, S.RBS, W.RBS or the S.promoter. To compensate for this, we used gel extractions dated from 15/8/2011 in order to complete ligations. These were as follows: OmpF (#1, #2, #3) + S.RBSOmpF (#1, #2, #3) + W.RBSRFP + TTW.promoter + S.RBS

These were then transformed into Top10 cells. Minipreps of the Promoter-YFP overnights made yesterday.one overnight per plate was selected and digested with EcoRI-PstI to check for size of the insert.the restriction digest didn’t work as expected for most of the samples, but worked for OmpF-sYFP and OmpF-wYFP.1ul of each overnight was pipetted onto a microscope slide and visualised under a fluorescence microscope (filter 1: 470-490 nm). pictures were taken.

25/08: Yesterday’s transformations returned poor growth initially, yet after a further couple of hours, growth was substantial enough to make o/n’s. Digests and ligations were created from various promoter (pBAD, Bluf, S.promoter, W.promoter) and RBS (Weak and Strong) constructs, as well as RFP. The eventual aim was to yield the following ligations: pBAD+W.RBS+RFPpBAD+S.RBS+RFPBluf+W.RBS+RFPBluf+S.RBS+RFPS.promoter+W.RBS+RFPS.promoter+S.RBS+RFPW.promoter+S.RBS+RFP These ligations were then transformed and left overnight. the same digests leftover from yesterday’s check gel were ran on a gel again.the high [DNA] in one of the digested samples prevented us to clearly see all the bands on the gel.Digestion of the biobricks sent over from the edinburgh team:- pLac-Phycocyanobilin pathway (EDIN 1) ---------------> SpeI-PstI- RBS-Cph8 (EDIN 2) -------------------------------> XbaI-PstI- Cph8-pOmpC-eYFP (EDIN 4) -----------------------> XbaI-PstIthe digestion didn’t work very well. low [DNA] and failure of the Restriction Enzymes postponed this digest.

26/08: Yesterday’s transformations yielded white colonies on 2 plates- a disappointing result. It was decided a new approach should be taken in an effort to ligate RFP to our Promoter/RBS constructs; after digesting with X/P and E/S respectively, both inserts will be ligated into a Tetracycline-resistance plasmid. However, even this attempt failed to return any positive results. concentrations of DNA were adjusted and 1ul of RE was used to repeat yesterday’s digest of the EDIN biobricks.we extracted from this gel what we believed to be the inserts and the following ligations were

performed:EDIN1 + EDIN2EDIN1 + EDIN4

Transformation of B-carotene and lycopene failed, so these were repeated.OmpC-sYFP and OmpF-sYFP were transformed into ΔEnvz CP919 chemically competent cells.

29/08: Yet more digestions and ligations were carried out in the search for a construct containing RFP. The Promoter+RBS constructs were cut with AlwnI/SpeI, whilst all RFP/RFP+TT constructs were cut with AlwnI/XbaI. It should be noted that two samples of RFP were cut with AlwnI and SpeI, and two samples of Double Terminator were cut with AlwnI and SpeI, so as to ligate these together to ensure there is definitely a supply of RFP+TT. Ligations that were carried out include: OmpC+W.RBS #1+RFP+TT #1OmpC+W.RBS #1+RFP+TT #2OmpC+W.RBS #2+RFP+TT #1OmpC+W.RBS #2+RFP+TT #2OmpF+S.RBS+RFP+TT #1OmpF+S.RBS+RFP+TT #2OmpF+W.RBS+RFP+TT #1OmpF+W.RBS+RFP+TT #2 These were then transformed into Top10 cells and left overnight. Digestion of Bluf-wYFP miniprep with EcoRI-PstI.all the 3 digested minipreps didn’t contain the insert.

30/08: Digested the following constructs with AlwnI and SpeI: Bluf+S.RBSW.promoter+S.RBSS.promoter+W.RBSBluf+W.RBS Subsequently ran these digests on a gel for visualization, however no bands could be seen. We also set up ligations of: OmpC+W.RBS #1+RFP+TT #1OmpC+W.RBS #1+RFP+TT #2OmpC+W.RBS #2+RFP+TT #1OmpC+W.RBS #2+RFP+TT #2OmpF+S.RBS+RFP+TT #1OmpF+S.RBS+RFP+TT #2OmpF+W.RBS+RFP+TT #1

OmpF+W.RBS+RFP+TT #2 All of which were transformed into Top10 cells. Digest #1: wYFP and sYFP with XbaI-PstI Bluf promoter with SpeI-PstIbands were cut and extracted but not ligated.

Digest #2: pBAD-sYFP I,II and III with EcoRI-PstI Bluf-sYFP IIIdone to check for any eventual success.no success was detected. no pBAD-YFP nor Bluf-YFP fragments showed up.

o/n of EDIN1+2 (Cm) Lycopene K274120 (Kn) transformations of EDIN1+4 (Cm) beta-carotene (Kn) pUC19 as +ve controlMinipreps and Glycerols of OmpF-sYFP and OmpC-sYFP.

31/08: Growth was observed on all transformations from the 30th! Overnights were made of all.exactly the same protocol was followed to repeat digests #1 and #2 from yesterday.Bluf and YFP fragments extracted the day before and left in the fridge overnight were ligated together and transformed in Top10 cells. these were then plated on ampicillin agar.Overnights of b-carotene, EDIN1+4, OmpC-sYFP and OmpF-sYFP were made.

01/09: Began digests of the following constructs: [A] OmpC+W.RBS #2 +RFP+TT #1[B] OmpC+W.RBS #2 +RFP+TT #2[C] OmpC+W.RBS #2 +RFP+TT #3[D] OmpF+S.RBS #1 +RFP+TT #2[E] OmpC+W.RBS #2 +RFP+TT #2[F] OmpC+W.RBS #1 +RFP+TT #2[G] OmpF+S.RBS #1 +RFP+TT #2[H] OmpC+S.RBS #2 +RFP+TT #3[I] OmpF+S.RBS #1 +RFP+TT #3[J] OmpC+S.RBS #1 +RFP+TT #2[K] OmpF+W.RBS #1 +RFP+TT #1[L] OmpF+W.RBS #1 +RFP+TT #2[M] OmpF+W.RBS #1 +RFP+TT #3[N] OmpC+S.RBS #1 +RFP+TT #1[O] OmpC+S.RBS #1 +RFP+TT #3[P] OmpC+W.RBS #2 +RFP+TT #2[Q] OmpC+W.RBS #2 +RFP+TT #2

We decided to digest them overnight and run the gel the following day. OmpF-sYFP and OmpC-sYFP ΔEnvz cells were made chemically competent and were then transformed with EDIN 1+2 and GlrN.EDIN 1+2 (II and III) and GlrN (I, II and III) were digested with EcoRI and PstI to check sizes of plasmids and biobrick inserts.EDIN 1+2 sizes didn’t match the expectations...GlrN looks ok.

02/09: It was noted that on the gel, only 6 digests from the 1st (E,H,I,J,M and O) have been successful. These were ligated into the submission vector and transformed into EnvZ negative cells. However, due to an accident, cells were added directly to ligation tubes, resulting in a final volume of 35μl (10μl DNA + 25μl cells). We finally decided to aliquot this volume to yield one 20μl solution and one 15μl solution, and to add 30μl and 35μl of LB respectively to yield 50μl in each. Each transformation therefore has two rounds; one designated “20μl”, the other “15μl”. Remade competent cells and transformed same as yesterday.More fluorescent microscopy.Minipreps of beta-carotene.

05/09: Minipreps of OmpC-strong RBS, OmpF-sRBS, pBAD-sRBS, Bluf-sRBS, sProm-sRBS, wProm-sRBS and pSB1C3 were digested with EcoRI-AlwnI to check for success of the ligation.

the relevant insert bands were extracted to be ligated onto the submission vector pSB1C3.unfortunately only OmpF-sRBS and the pSB1C3 showed up on the gel, thus only that ligation was attempted on the following day (6th August).

minipreps from the successful o/n of Bluf-sYFP, Bluf-wYFP, OmpC-sYFP and EDIN1+4 were digested with EcoRI-PstI to check the size of the inserts.the gel results indicate that only Bluf-wYFP contains the correct insert between the suffix and prefix.

06/09: Growth was happily observed on all transformation plates, except: E+pSB1C3 (15μl)E+pSB1C3 (20μl)J+pSB1C3 (15μl) From the ones that did work, o/n’s were made. the ΔEnvZ cells containing OmpC-sYFP, OmpC-sRFP, OmpF-sYFP and OmpF-sRFP were all made chemically competent to introduce the plasmid encoding the light receptors in the respective cells; for example the green light receptor GlrN was transformed into ΔEnvZ-OmpC-sYFP and -RFP cells. This way we are going to obtain light-responsive E.Coli that will report any light absorption with YFP and RFP.

ΔEnvZ competent cells were transformed with the light receptors EDIN1+2, EDIN1+4 and GlrN.ΔEnvZ-OmpC-sYFP was transformed with GlrN,

ΔEnvZ-OmpF-sYFP was transformed with EDIN1+2,ΔEnvZ-OmpC-sRFP was transformed with GlrN,ΔEnvZ-OmpF-sRFP was transformed with EDIN1+2,

and each competent cell type was also transformed with pUC19 as a +ve control.The controls were plated on Carbenicillin agar and x-gal to help detect YFP expression. Since these cells were prepared by our lab, we changed the protocol to increase the success of transformation: 50ul of comp. cells were used for each reaction, together with 1ul of DNA. The digested pSB1C3 and OmpF-sYFP fragments from yesterday’s gel were kept in their cut slice of gel, in an eppendorf, overnight in the fridge. these were then extracted and a single ul of each gel extract was run on a new gel to check for the concentration before attempting the ligation.the fragment containing OmpF-sYFP was so faint that didn’t show up on the gel at all.the ligation plan was aborted for lack of reagent DNA. 07/09: Minipreps were created from each o/n. From here, we created digests of the following using EcoRI and PstI: H + pSB1C3 (20μl) I + pSB1C3 (15μl)O + pSB1C3 (15μl)M + pSB1C3 (15μl)M + pSB1C3 (20μl)J + pSB1C3 (20μl)O + pSB1C3 (20μl) Both O (OmpC+S.RBS +RFP+TT) and J (OmpC+S.RBS +RFP+TT) lanes returned visible bands of desired length, meaning that the presence of these full constructs is likely. miniprepped Bluf-sYFP and Bluf-wYFP (3 each) were all digested with EcoRI-PstI to check for size of insert. Bluf-wYFP #1 and Bluf-sYFP #3 showed the expected band pattern on the gel. The GlrN transformation of the past days didn’t produce any colony so the transformation was repeated again. GlrN was introduced into chemically competent ΔEnvZ-OmpC-sYFP and -sRFP. O/ns of the successful ΔEnvZ-OmpF-sYFP containing EDIN1+2 were used to draw our DISColi logo on selective agar plates. Each of the o/n was used to draw to logos, one of them was then wrapped in aluminium foil and both were then incubated at 37’C overnight. 08/09: Transformation of Glrn into competent ΔEnvZ-OmpC-sYFP and -sRFP was repeated, due to the failure of the previous ones. a whole new set of overnight was made: 3 x ΔEnvZ-EDIN1+4 3 x ΔEnvZ-EDIN1+2 3 x ΔEnvZ-OmpF-sYFP-EDIN1+2 3 x ΔEnvZ-OmpF-sRFP-EDIN1+2

1 x ΔEnvZ-OmpC-sRFP-GlrN (probably contamination)

Samples from older overnights were observed under the fluorescent microscope and pictures were taken.

09/09: the overnights set up yesterday were miniprepped and digested with EcoRI-PstI and ran on a 1% gel. The gel made clear that many of our constructs are wrong, the OmpF-sYFP ones are alright, but the EDIN1+2 and EDIN1+4 light receptors don’t look right. More microscopy of over-nighted samples was carried out. 12/09: GlrN was transformed into ΔEnvZ-OmpC-sYFP and -sRFP cells once again. this time all the possible different competent cells were used and all the different minipreps of GlrN (all 3). 14/09: o/n of the transformation from monday 12th september. these were done in duplicates and one for each plate was wrapped in aluminium foil before incubation at 37’C.