IGEM:Imperial/2010/Surface Protein team


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Useful resources (Click me) Main Page Parasite Detection Surface Protein</li> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/XylE_team" class="popt">XylE team</a></li> <a href="http://www.openwetware.org/wiki/IGEM:Imperial/2010/Vectors_team" class="popt">Vector team</a></li> </ul>

Follow our progress (Click me) <a href="#Week_6" class="popt">Week 6</a></li> <a href="#Week_7" class="popt">Week 7</a></li> <a href="#Week_8" class="popt">Week 8</a></li> <a href="#Week_9" class="popt">Week 9</a></li> <a href="#Week_10" class="popt">Week 10</a></li> <a href="#Week_11" class="popt">Week 11</a></li> <a href="#Week_12" class="popt">Week 12</a></li> </ul>


 * The aim of our team is to assemble the 6 different versions of lytC with the AIP attached to it. For this we first have to use PCR to get lytC out of the genome of B. subtilis whilst adding a RBS using primer extension. Then we will add a promoter as well as 6 different C-termini with linker, signal (AIP) and tag sequnces. Finally we will move these constructs into a new vector to which we have added a terminator.

Friday, 13th-Aug-2010


Plan:


 * Gel to check if pSB1C3 PCR worked. (Between EcoRI site and PstI site)
 * If PCR worked we can purify pSB1C3
 * Start digestion with restriction enzymes.
 * If possible we want to set up ligation over the weekend

Report:
 * As expected we observed a 2kb fragment on the gel. This confimrs that the PCR of pSB1C3 was successful as the vector is approximately 2kb long.
 * Having confirmed that the PCR was successful we purified the PCR product from the solution using the E.Z.N.A.® Cycle Pure Kit (Omega bio-tek) but used 25µl of ddH2O instead of elusion buffer in the last step.
 * We then did a restriction digestion for:
 * 1) The pSB1C3 PCR product using the enzymes PstI and EcoRI in buffer 4
 * 2) B0014 (the terminators) using the enzymes PstI and EcoRI also in buffer 4


 * Our last step was running the digestion product on a gel to confirm successful digestion. Unfortunately we lost the digested BOO14 during electrophoresis so digestion of BOO14 and electrophoresis have to be repeated.

Saturday, 14th-Aug-2010
We had to repeat some of the steps from the 13th.

Plan:
 * Repeat digestion of the terminator (BOO14)
 * Run gel of the digestion products to isolate terminator

Report:
 * Digestion of terminator (on pSB1AK3) with PstI and EcoRI
 * Gel confirms that digestion was successful, although yield appears to be relatively low

Monday, 17th-Aug-2010
Plan:
 * Gel purify terminator (BOO14)
 * Ligate vector (pSB1C3) and terminator.

Report:
 * We successfully purified BOO14 from the gel using the QIAquick® Gel Extraction Kit (250) but used 50µl of ddH2O instead of elusion buffer in the last step. Then we determined its concentration as well as the concentration of pSB1C3 by running a gel and comparing the intensity of the bands with the 1kb ladder.
 * Having determined the rough concentration of our DNA we set up over-nigh ligation of the two. We used two different ratios of pSB1C3 to BOO14: 0.5µl:3.5µl and 1µl:3µl. As the final concentration of the ligation product is very low we will transform E. coli to be able to analyse bigger quantities of the vector at a later point.

Tuesday, 18th-Aug-2010
Plan:
 * Transformation of E. coli with the ligation product.
 * Update Wiki

Report:
 * E. coli was transformed using chemical competence and heat shock. We prepared plates with our transformants to be incubated overnight.
 * We reorganized and updated our Lab-Page on the Wiki, including new tables, upload of pictures and result as well as user-interface optimisation.

Wednesday, 19th-Aug-2010
Plan:
 * Confirm successful transformation by colony PCR
 * Set up replica plates
 * Prepare culture in LB for Midi-Prep

Report:
 * The PCR indicates that our ligation and transformation were successful, however due to contamination, as demonstrated by our negative control, we will have to repeat the colony PCR on Thursday. We set up an additional PCR to test the individual components of our PCR reaction to find out which one is contaminated with DNA.

Primers have not arived yet

Thursday, 20th-Aug-2010
Plan: If primers have arrived:
 * Run a gel of previous day's PCR to find out which PCR reagent is contaminated.
 * Miniprep to get pSB1C3 with BOO14
 * Run PCR to amplify the cell wall binding (CWB) domain of Lyt C from B. subtilis.
 * Electrophoresis of PCR product to confirm correct amplification
 * PCR purification of CWB from solution

Report:
 * We found that the Barnes buffer was contaminated.
 * Repeated the SCP, and observed a band at 100bp, which indicates that the ligation was successful, and the B0014 part (the terminators) is present in the plasmid, pSB1C3.
 * To make sure that this was the case, we digested the miniprepped plasmids with EcoRI and SpeI. However, there was only a very faint band on the gel at 100bp.

Friday, 21st-Aug-2010
Plan:
 * Repeat a gel of digested plasmids, but run for only 10 minutes.
 * If primers have arrived, PCR the LytC cell wall binding domain.

Report:
 * The repeated gel showed only a very faint band at 100bp, so we can't be completely sure that the ligation was successful.
 * Therefore we carried out a restriction digest with AseI (which cuts within B0014) and NcoI (which cuts within pSB1C3).
 * When we run the restriction fragments on a gel, we will know that the ligation is successful if there is a band at 1324bp and 843bp.


 * We also did a PCR to get the LytC cell wall binding domain out of the Bacillus genome.

Sunday, 22nd Aug 2010


Plan:
 * Run a gel to see if:
 * 1) The PCR of the LytC cell wall binding domain worked
 * 2) The digestion with AseI and NcoI gave fragments that correspond to a successful ligation. This is because AseI will only cleave if the B0014 part ligated successfully into the vector.

Report:
 * The gel showed that:
 * 1) The ligation definitely worked! The correct fragment sizes were observed on the gel.
 * 2) The primers in the PCR may have annealed nonspecifically, because the PCR product did not resolve properly in the gel.


 * To prepare the ligated plasmids for midiprep tomorrow, overnight cultures were set up using 100ml LB (containing chloramphenicol), which was innoculated with cells from the original replica plates.


 * A second PCR was set up with a gradient of increasing annealing temperatures, which would hopefully ensure specific binding of primers. (We used taq in this case, just to see which temperature gave us the best result)
 * The gel of the PCR products showed that all 3 PCRs were successful. So we can now use Pfu in the next PCR to obtain the LytC cell wall binding domain.

Monday, 23rd Aug 2010
Plan:
 * Run a PCR of the LytC CWB domain using Pfu and altered annealing temperatures.
 * If PCR is successful, digest with DpnI to remove template DNA
 * Gel purify LytC CWB domain
 * Midi-prep plasmids (pSB1c3 ligated w/ B0014)

Report:
 * The PCR was successful and the LytC CWB domain was gel purified (after DpnI digest)
 * The plasmids were miniprepped, and the final DNA concentration was 120ng/µl
 * Overnight digestion of lytC with SpeI was set up. XbaI will be added to the mixture in the morning making the final volume of the digestion 30µl

Tuesday, 24th-Aug-2010
Plan:
 * Add XbaI to digestion and incubate for 60+ min
 * Ligation of lytC with pSB1C3

Report:
 * Due to problems our midi-prep of pSB1C3, we only digested the two components but then had to set up another culture of B. subtilis for midi-prep

Wednesday, 25th-Aug-2010


Plan:
 * Midi-Prep to get pSB1C3 with BOO14 from cultur set up yesterday
 * Just electrophoresis to check for correct midi-prep product
 * Perform XbaI + SpeI digestion to confirm the terminator is in the plasmid
 * Gel purify digestion product and test small portion on gel

Report:
 * Our gels indicate that the terminator is in the vector and that the midi-prep was successful this time

Thursday, 26th-Aug-2010
Plan:
 * Determine relative concentrations of pSB1C3 and lytC after digestion
 * Dephosphorylation of plasmid
 * Set up ligation of pSB1C3 with lytC (overnight and bench)
 * Use bench ligation for transformation and plate E. coli
 * Set up PCR to get promoter out of vector (test of best temperature with taq)
 * Amplify promoter (2 identical samples) by PCR using Pfu for high fidelity
 * PCR purification of on pVEG sample
 * Gel electrophoresis of PCR product (one sample alsoPCR purified) pVEG

Report:
 * We found that pSB1C3 had a about 4 time higher concentration than lytC.
 * We set up a 10µl dephosphorylation reaction with 2µl of pSB1C3 and incubated for 10min.
 * 2.5µl of the dephosphorylation reaction were used together with 2µl of lytC for a 10µl ligation reaction. This was split into a 5µl overnight ligation and a 5µl bench ligation reaction.
 * After 2 hours incubation at room temperature we transformed E. coli with the ligated vector and later plated it.
 * We set up a test PCR using taq polymerase to determine the best protocol to get the promoter pVEG out the current vector (pSB1AK3?).
 * Gel electrophoresis of the PCR products suggested that the following protocol was most suitable:
 * 1) 5 cycles at 60°C
 * 2) 25 cycles at 66°C
 * We set up another PCR reaction using Pfu for high fidelity with two identical samples
 * Sample 1 was later PCR purified by Kirill and Kyascha (thanks guys!)

Friday, 27th-Aug-2010


Plan:
 * Gel electrophoresis of both PCR sample of pVEG to find out if we can use the PCR purified sample (1) or have to gel purify sample 2. This is due to the small size of pVEG which might make it impossible to PCR purify it.
 * Check if transformation was suucessful. If yes, set up a midi-prep. If not, use overnight ligation transform again
 * Digest pVEG (promoter)EcoI * XbaI
 * Gel electrophoresis to determine if digestion was successful
 * Depending on gel either gel or PCR purification of pVEG.

Report:
 * Both samples of pVEG were successfully puified and can both be used in the following cloning steps
 * Our transformation was unsuccessful and no background was observed either. We thus used our overnight ligation to transform E. coli
 * We decided to digest the promoter with EcoRI + SpeI rather than EcoRI + XbaI. Digestion with SpeI was set up overnight but the digestion volume was accidentially made up to a total of 30µl (rather than 28.5µl allowing for EcoRI to be added the next day)

Saturday, 28th-Aug-2010
Plan:
 * Check if transformation of E. coli with overnight ligation of pSB1C3 with lytC was successful
 * Add EcoRI to digestion of pVEG (E+S) and incubate
 * Analyse digestion using gel electrophoresis

Report:
 * Transformation of E. coli using our overnight digestion was unsuccessful which means we have to restart construction of our lytC vector
 * EcoRI was added to the pVEG digestion and both samples were incubated for 1 hour
 * 5µl of sample 1, which is to be PCR purified later, were loaded on a gel and 30µl (all) of sample 2, which is to be gel purified. The electrophoresis confirmed that pVEG had been digested and the bands of smaple 2 (two lanes in total) were cut out of the gel for gel purification (0.44g)

Monday, 30th-Aug-2010
Plan:
 * Gel Purify pVEG (digested E+S) sample 2
 * PCR purify pVEG (digested E+S) sample 1
 * Gel electrophoresis to check if purifications were successful
 * PCR of LytC for blunt-ended ligation
 * Digest gel purification of LytC with SpeI overnight

Report:
 * Both sample were purified successfully as the gel confirmed

Tuesday, 31st-Aug-2010


Plan:
 * Digestion of pSB1C3 EcoRI + SpeI
 * Gel electrophoresis of digestion product
 * Gel purification of pSB1C3
 * Gel electrophoresis to determine concentrations of insert and vector
 * Dephosphorylation of pSB1C3
 * Set up of overnight ligation

Report:
 * Electrophoresis confirmed that digestion of pSB1C3 with EcoRI and SpeI was successful and the terminator removed
 * The digested vector was later purified and the relative concentrations of the digested pSB1C3 and pEVG determined. Our samples contained about 30 times as much vector as promoter and since the vector is about 20 times larger than the insert we decided to ligate overnight in a ration of 1.5 volumes of pVEG to 1 volume of dephosphorylated vector.

Wednesday, 1st-Sep-2010


Plan:
 * Digestion of pSB1C3-B0014 with XbaI and SpeI. This will then be gel purified and dephosphorylated.
 * XbaI was added to the SpeI digest of LytC. This will then be PCR purified.
 * A ligation reaction of LytC into pSB1C3 will be set up at the end of the day.

Report:
 * All planned steps worked and overnight ligations were set up.

Thursday, 2nd-Sep-2010
Plan:
 * Measurment of 5 midi-preps using a spectrophotometer using water for callibration.
 * Digestion and incubation of the midi-preps.
 * Gel purification of digested DNA

Report:
 * The spectrophotometer caused great problem as it continuously, and even when operated by different people, produced wrong and inconsistent results as well as failing to remain callibrated.
 * We therefore used gel electrophoresis to determine the sample concetrations (2 lanes each, 1 and 5ul).
 * Gel electrophoresis also yielded bad results so we will measure concentrations tomorrow using a different method.
 * Neither digestion nor purification were performed.

Friday, 3rd-Sep-2010
Plan:
 * Because two different methods of determining the concentration of our 5 Midi-Preps failed yesterday, we will use a specrophotometer in the Biochem building today
 * Updates to the Wiki Protocol page as well as our Lab page
 * Check for successful transformations
 * If we have transformants:
 * 1) Make a replica plate
 * 2) Perform a colony PCR
 * 3) Gel electrophoresis to analzye colony PCR results

Report:
 * We measured the concentration of DNA in the midi-preps (of plasmids containing synthesis products). The concentrations varied between 78ng/ul and 230ng/ul (one will have to be repeated as the yield was too low).
 * The transformation of the LytC-pSB1C3 was successful. A colony PCR was done and a replica plate set up.
 * The gel did not confirm that the ligation was successful, so another gel will need to be run on Monday.

Monday, 6th-Sep-2010
Plan:
 * Repeat of gel for 7th colony PCR, because it showed a faint band around 1kb, indicating that LytC was present in the vector.
 * 20 further colony PCRs from the LytC transformants.
 * Set up miniprep cultures from (a) blunt-ended ligation and (b) numbers 5, 7, 18 and 19 from the original colony PCR.

Report:
 * All of the planned steps were carried out.
 * The second colony PCR products were run on a gel, but we think the primers may not be annealing because the bands at 1kb were not observed.

Tuesday, 7th-Sep-2010
Plan:
 * Minipreps from overnight cultures.
 * Restriction digest (XbaI & SpeI) to see if;
 * 1) The LytC cell wall binding domain is in the plasmid
 * 2) The insert is in the correct orientation
 * Gel electrophoresis to analyse digestion

Report:
 * Plasmids from overnight cultures were successfully miniprepped.
 * The uncut and cut plasmids (using XbaI and SpeI) were run on a gel.

Wednesday, 8th-Sep-2010
Plan:
 * Start the repeat of the pVeg transformation
 * Repeat the restriction digest of miniprepped LytC plasmids using XbaI and SpeI as well as just XbaI on its own for Kirsten's plasmids and EcoRI and SpeI as well as EcoRI only for the pSB1C3 vector made by us.

Report:
 * pVEG was amplified from its vector by PCR
 * Gel electrophoresis confirmed that PCR was successful although a big, strong band appeared far above the expected length of pVEG as well
 * pVEG was gel purified
 * pVEG was digested with SpeI ovenight
 * The repetition of the restriction digest was done but appareantly failed ''(it turned out later that one of the settings of the UV-box had been changed so not enough light was detected)
 * Another digestion was started but will be finished tomorrow

Thursday, 9th-Sep-2010
Plan:
 * Continue digestion of mini-preps with XbaI/EcoRI with and without SpeI
 * Addition of XbaI to the overnight digestion of pVEG with SpeI, then PCR purify
 * Digestion of vector pSB1C3 and gel purify
 * Set up ligation of pVEG and pSB1C3
 * Set up overnight cultures so tomorrow we can miniprep plasmids containing the LytC cell wall binding domain

Report:
 * pVEG seems to have disappeared, so a repeat PCR was done, followed by PCR purification. This was then used for an overnight digestion of SpeI. Unlike last time, the gel purification step was missed because it decreased our yield too much.
 * Therefore ligation of pVEG and pSB1C3 was not done
 * Digestion of lytC and subsequent gel analysis showed a possible candidate from Kirsten's blunt ended ligations (K5)
 * Midi-Prep culture for K5 was set up
 * 23Mini-Prep cultures from our ligation plate were set up
 * A test culture was set up to check new Chloramphenicol works

Friday, 10th-Sep-2010
Plan:
 * Miniprep using overnight cultures (23 x LytC-pSB1C3 from our ligation)
 * Midi-Prep K5
 * Repeat of digest of pVeg

Report:
 * pVeg was set up in a restriction digest with SpeI
 * The over-night culture of K5 failed because the wrong antibiotic was used. A new culture was set up with the correct antibiotic
 * 23 Mini-Preps of lytC-pSB1C3 ligation were prepared and a restriction digest was prepared for the next day

Saturday, 11th-Sep-2010
Plan:
 * Add EcoRI to pVEG restriction digest
 * XbaI and SpeI restriction digestion of 23 Mini-Prep
 * Gel electrophoresis of all digestions
 * Cut out pVEG for Gel purification
 * Midi-Prep of K5

Report:
 * EcoRI was added to the pVeg restriction digest
 * Gel electrophoresis of the restriction digest of the mini-preps indicated that restriction did not work as expected, maybe because one of the enzymes did not cut as a result of a wrong insert
 * pVEG was cut out of the gel to be gel purified
 * K5 Midi-Prep was started and paused after step 13 of the protocol

Sunday, 12th-Sep-2010
Plan:
 * Another diagnostic digest of the mini-preps with EcoRI and SpeI and EcoRI only
 * Gel purification of pVEG
 * Dephosphorylation of pSB1C3ation of pVEG
 * Ligation of pVEG and pSB1C3
 * Midi-Prep of Kirsten's lytC (K5)
 * Set up 3 Mini-Prep cultures of K5

Report:
 * Digested pVeg was gel purified and ligated with digested, dephosphorylated pSB1C3
 * Restriction digest showed that SpeI did not cut the vector
 * The Midi-Prep was tested for DNA concentration which turned out to be close to 0
 * 3 Mini-Prep cultures for K5 were set up

Monday, 13th-Sep-2010
Plan: Report:
 * 3 Mini-Preps of K5
 * Restriction digest of K5 and pSB1C3 with XbaI and SpeI
 * Transform E. coli with pVeg ligations
 * Gel purification of digested K5 and pSB1C3
 * Gel electrophoresis to determine concentrations of digests
 * Dephosphorylation of pSB1C3
 * Ligation of pSB1C3 overnight
 * The Mini-Preps were successfully prepared and two of them were used for a digestion with XbaI and SpeI. The pSB1C3 with B0014 vector was also digested with XbaI and SpeI which cut out the terminator B0014
 * Gel electrophoresis was used to confirm correct restriction and the bands were then gel purified
 * After gel purification the relative concentration of vector to insert were determined by gel electrophoresis
 * After dephosphorylation of pSB1C3 overnight ligation was set up
 * E. coli were transformed with pVEG but could not be plated so transformation will have to repeated

Tuesday, 14th-Sep-2010
Plan:
 * Meet Professor Alan Fenwick OBE, Director of the Schistosomiasis Control Initative (SCI)
 * Transform E. coli with
 * 1) the ligated pSB1C3 with lytC (from K5 culture)
 * 2) the ligated pSB1C3 with pVeg


 * Repeat PCR of pVeg with a shorter extension time, so that the vector is not also amplified.

Report:
 * Transformations were done as described above.
 * The meeting with Prof Fenwick and Dr Harrison of the SCI was very successful. Feedback can be found on the Human Practices page.
 * The PCR of pVeg with the shorter extension time produced a much higher concentration of pVeg in relation to pSB1C3.

Wednesday, 15th-Sep-2010
Plan:
 * If transformations were successful, carry out a colony PCR and set up overnight cultures for minipreps.
 * Gel electrophoresis to analyse PCR
 * Wiki-Meeting

Report:
 * Transformations of E. coli with pVEG and lytC was successful

Thursday, 16th-Sep-2010
Plan:
 * Mini-Prep of LytC (7) and pVEG (4) cultures
 * Digestion with
 * 1) XbaI and SpeI
 * 2) AccI
 * Gel electrophoresis of digest

Report: The Mini-Preps of LytC were done and then digested with XbaI and SpeI for the double digest, as well as AccI for the single digest, however Mini-Preps of pVEG failed, because I made a mistake and will be repeated tomorrow (cultures hav been set up). Unfortunately the digestion of LytC were not conclusive because Either XbaI or SpeI did not cut, nor did AccI. Therefore the digest will be repeated tomorrow with EcoRI rather than XbaI

Friday, 17th-Sep-2010
Plan: Report:
 * New Mini-Preps for pVEG and the Linkers
 * Restriction digest of pVEG in pSB1C3, LytC in pSB1C3 and Linkers in pSB1C3 with EcoRI and SpeI
 * Gel electrophoresis of digest
 * Mini-Preps were successfully made
 * The digestions showed that lytC in most likely not in the vector

Monday, 20th-Sep-2010
Plan:
 * Prepare K5M3 (lytC in blunt ended ligation vector) as well as H2 and H3 for sequencing
 * Digestion of H2 and H3 with AseI
 * Gel electrophoresis of digest
 * Prepare replica plate and mini-prep cultures for Gly-X Com AB

Report:
 * Samples were send of for sequencing. No primer had to be added to K5M3 because it is avaliable at the company
 * AseI did not cut either sample of pVEG which is a positive result because AseI does not cut pEVG but the terminator, allowing us to say with some confidence that we have got pVEG. Sequencing will give us certainty soon
 * Replica plate and mini-prep cultures for Gly-X Com AB were set up

Tuesday, 21st-Sep-2010
Plan:
 * Mini-prep of Gly-X Com CDE 1-2
 * Restriction digest of the linkers with EcoRI and SpeI as well as AccI
 * Gel electrophoresis
 * Set up Midi-Prep cultures for H2 and H3 (pVEG in pSB1C3)

Report:
 * Many, but not all of the samples, appear to have worked well. HEL-TEV 1-3 have worked, Gly-X Com CDE 2-3 have worked, Flex Com CDE has failed so far, but more mini-prep samples will be tested tomorrow, Flex TEV 2 worked, and both Gly-X Com AB 1-2 have worked
 * Culture for H2 and H3 were set up

Wednesday, 21st-Sep-2010
Plan:
 * If sequencing for H2 and H3 confirmes that pVEG is in the vector: Midi-Preps
 * Determine concentration of DNA in Midi-Prep
 * Test of further Mini-Preps of linkers (Flex Com CDE) with EcoRI+SpeI and AccI
 * Gel electrophoresis of digests

Report:

Thursday, 22nd-Sep-2010
Plan: Report:
 * Determine concentration of DNA in Midi-Prep (pVEG)
 * Set up Midi-Prep cultures of the linkers that have been successfully mini-preped
 * Set up mini-prep cultures was well as a replica plate of Gly-X Com AB
 * Concentration of DNA in H2 was ~950ng/µl
 * Concentration of DNA in H3 was ~430ng/µl
 * All cultures were set up and replica plate of Gly-X Com AB made

Friday, 23rd-Sep-2010
Plan: Report:
 * Mini-Prep of ComD from the cultures set up yesterday
 * Midi-Prep of the linkers from the cultures set up yesterday
 * Restriction digest of ComD Mini-Preps with EcoRI and SpeI
 * Measure Midi-Prep DNA concentration
 * ComD was mini-preped successfully and the restriction digest confirmed that it is ComD
 * The Midi-Preps of the linkers Hel-TEV, Flex TEV, Gly-X ComCDE and Gly-X ComAB were made and the concentration determined.