iGEM 2019 — Team Pittsburgh

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# Team:Pittsburgh/Notebook

[Pitteins](/Team%3APittsburgh)

[Team](/Team%3APittsburgh/Team)

[Project](/Team%3APittsburgh/Description)

[Modeling](/Team%3APittsburgh/Model)

[Human Practices](/Team%3APittsburgh/Human_Practices)

[Safety](/Team%3APittsburgh/Safety)

[Team](/Team%3APittsburgh/Team)

[Attributions](/Team%3APittsburgh/Attributions)

[Collaborations](/Team%3APittsburgh/Collaborations)

[Description](/Team%3APittsburgh/Description)

[Design](/Team%3APittsburgh/Design)

[Parts](/Team%3APittsburgh/Parts)

[Experiments](/Team%3APittsburgh/Experiments)

[Notebook](/Team%3APittsburgh/Notebook)

[Contribution](/Team%3APittsburgh/Contribution)

[Results](/Team%3APittsburgh/Results)

[Human Practices](/Team%3APittsburgh/Human_Practices)

[Public Engagement](/Team%3APittsburgh/Public_Engagement)

## Notebook

## Week 3

Wed (5/29/2019) Took inventory of lab

Ripal, Jemy, Victor

Wed (5/29/2019) Autoclaved pipette tips & eppendorf tubes

Jemy, Victor

Fri (5/31/2019) Autoclaved LB & LB agar

Victor, Jemy, Ripal

Fri (5/31/2019) Transformed E. coli with plasmids pBAD02 and pskDuet1

Victor

Plasmids obtained from graduate student Dylan Tomares in the Childer's lab.

Sat (6/1/2019) Miniprepped plasmids

Victor

Sat (6/1/2019) Colony PCR

Victor

Sat (6/1/2019) Started liquid cultures

Victor

(PBAD02 Ampicillin, pskDuet1 Kanamycin, other pTEV Ampicillin)

Sat (6/1/2019) Created freezer stock for pBAD02, pskDuet1

Victor

## Week 4

Sun (6/2/2019) Linearized pTEV backbone

Victor

![](/wiki/images/2/20/T--Pittsburgh--data_week4_pTEV_Gel.jpg)

## Week 6

Wed (6/19/2019) Received set 1 gBlocks from IDT

\*

Wed (6/19/2019) Cloned set 1 gBlocks into pTEV5 and transformed DH5α cells

Victor

![](/wiki/images/4/4d/T--Pittsburgh--data_week6_6.20.2019_Gibson_Plates.jpg)

DH5α colonies following Gibson assembly

Thu (6/20/2019) Colony PCR of set 1 constructs

Victor

![](/wiki/images/2/26/T--Pittsburgh--data_week6_pcr_1.jpg)

Colony PCR - 1

Top: pVS28:(3,4,5,6,7,8,1), pVS29:(1,2,3,4), pVS43:(1,2,3,4), pVS29:(5)

Bottom: pVS29:(6), pVS30:(1,2,3,4,5,6), pVS31:(1,2,3,4,5,6,7,8)

![](/wiki/images/d/d7/T--Pittsburgh--data_week6_pcr_2.jpg)

Colony PCR - 2

Top: pVS34:(1,2,3,4,5,6,7,8), pVS33:(1)

Bottom: pVS33:(2,3,4,5,6,7,8), pVS28:(1,2)

Fri (6/21/2019) Made DH5α & BL21 comp cells

Victor, Harrison

Fri (6/21/2019) Miniprepped set 1 constructs to prepare for BL21 transformation

Victor, Harrison

Fri (6/21/2019) Transformed set 1 constructs into BL21 for expression

Victor, Harrison

![](/wiki/images/6/6f/T--Pittsburgh--data_week6_bl21_plates.jpg)

BL21 transformation plates

(slightly overgrown)

## Week 7

Sun (6/23/2019) Started liquid cultures of set 1 constructs in BL21

Victor

Mon (6/24/2019) Small scale expression test

all

![](/wiki/images/3/3c/T--Pittsburgh--data_week7_expr.jpg)

Expression gel (SDS-PAGE)

Left to right: pVS(28,29,30,31,33,34,43)

Our ladder was old and did not work

(we got better at running gels I promise)

Mon (6/24/2019) Prepared buffers for protein purification

Victor, Harrison

Tue (6/25/2019) Test purification of pVS33 with Ni-NTA affinity chromatography

all

Dylan Tomares, a graduate student from the Childer's lab assisted us with purification of pVS33.

![](/wiki/images/9/9c/T--Pittsburgh--data_week7_purification.jpg)

Purification gel (SDS-PAGE)

Insufficient lysis caused protein to be retained in the pellet

Tue (6/25/2019) Started overnight liquid cultures for miniprep

all

Wed (6/26/2019) Miniprepped cultures for sequencing

Ripal, Jemy, Mel, Harrison

Plasmid concentration was insufficient for Sanger sequencing.

Wed (6/26/2019) Started overnight liquid cultures for miniprep

Victor

Thu (6/27/2019) Miniprepped cultures and submitted for sequencing

Victor

Thu (6/27/2019) Expressed, induced and pelleted two 250mL cultures of pVS33

Victor, Harrison

Fri (6/28/2019) Attempted purification of pVS33

Victor, Harrison

Unable to lyse cells completely with sonic dismembrator.

## Week 8

Mon (7/1/2019) Sonic dismembrator tests

all

We tried several different cycle patterns on the sonic dismembrator to determine the best mode of lysis moving forward.

![](/wiki/images/1/13/T--Pittsburgh--data_week8_dismembrator.png)

Dismembrator gel (SDS-PAGE)

L=ladder, P=pellet, S=supernatant

* 1a - (10 sec on/20 sec off) x 20
* 1b - (10 sec on/20 sec off) x 40
* 2a - (15 sec on/15 sec off) x 20
* 2b - (15 sec on/15 sec off) x 40
* 3a - (25 sec on/5 sec off) x 15
* 4 - (1.5 min on)
* C - no lysis

Tue (7/2/2019) Expressed TEV protease and pVS43

all

## Week 9

Mon (7/8/2019) Small scale expression test

all

We attempted another small scale expression test. Cells were grown to OD600=0.5 and induced to 1mM IPTG for 4 hours at room temperature.

![](/wiki/images/0/09/T--Pittsburgh--data_week9_7_8_2019_expr.png)

Expression test gel (SDS-PAGE)

For each construct, left=uninduced, right=induced

Mon (7/8/2019) TEVp purification attempt

all

Starting with a 250mL culture of TEVp, we attempted Ni-NTA purification again using our new lysis protocol. Unfortunately, almost all of the protein was lost in the flow-through indicating poor his-tag binding or a faulty column/protocol.

![](/wiki/images/c/c0/T--Pittsburgh--data_week9_7_8_2019_tev_purification.png)

Purification test gel (SDS-PAGE)

M=marker, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Tue (7/9/2019) Transformation of set 2

Victor

We received cloned-in plasmids from Twist with out set 2 constructs and transformed them into BL21.

Wed (7/10/2019) Purification tests

Harrison, Jemy, Ripal

We took 20mL cultures of pVS29, pVS31 and pVS33 and attempted Ni-NTA purification. However, based on an A280 reading, we observed no protein in our elution -- likely due to excessive washing.

Fri (7/12/2019) Expression of pVS(29,31,33)

Harrison, Jemy, Ripal

We expressed 250mL cultures of constructs pVS(29,31,33) for further purification tests.

The lower band for each construct is our protein of interest and the top band is LacI.

![](/wiki/images/b/b2/T--Pittsburgh--data_week9_7_12_2019_expr.png)

Expression gel (SDS-PAGE)

For each, left=uninduced, right=induced

## Week 10

Tue (7/16/2019) Purification of pVS(31,33)

Harrison, Jemy, Ripal

We tried purification of pVS31 and pVS33. Unfortunately most of our protein was retained in the pellet indicating insolubility.

![](/wiki/images/e/ea/T--Pittsburgh--data_week10_purification.png)

Purification gel (SDS-PAGE)

P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

Tue (7/16/2019) Purification of pVS(31,33) with Emulsiflex

Victor

Attempted purification of pVS31 and pVS33 with an Emulsiflex. Most of the protein was retained in the pellet indicating solubility (as above).

![](/wiki/images/9/96/T--Pittsburgh--data_week10_purification_e.jpg)

Purification gel (SDS-PAGE)

## Week 11

Tue (7/23/2019) Small scale expression tests

all

Since our previous expression tests were still relatively uncertain, we tried incubating to a higher OD (0.5-0.6) and expressing for 5 hours at room temperature.

We expressed set 1 constructs pVS(28,29,30,31,33,34,43) as well as our newly transformed set 2 constructs pVS(20,22,23,24,25,26).

Everything expressed at the expected size except for pVS28 (which did not express). After looking back at the sequencing results, we realized that our Gibson assembly introduced a deletion in the reading frame leading to a frame shift mutation.

![](/wiki/images/7/73/T--Pittsburgh--data_week11_expr_20_26.png)

Expression gel (SDS-PAGE)

* pVS20: 32.02 kDa
* pVS22: 21.44 kDa
* pVS23: 14.90 kDa
* pVS24: 32.02 kDa
* pVS25: 13.62 kDa
* pVS26: 20.75 kDa

![](/wiki/images/9/9b/T--Pittsburgh--data_week11_expr_28_43.png)

Expression gel (SDS-PAGE)

* pVS28: 19.86 kDa
* pVS29: 18.80 kDa
* pVS30: 18.69 kDa
* pVS31: 24.86 kDa
* pVS33: 19.91 kDa
* pVS34: 21.85 kDa
* pVS43: 21.44 kDa

Wed (7/24/2019) Extract inserts via PCR

Victor

Since several of our constructs appeared to be relatively insoluble, we decided to try cloning our inserts into pTEV6 which expresses maltose binding protein as an N-terminal fusion tag.

To extract our insert, we ordered insertion primers and ran PCR followed by gel electrophoresis. Then we cut out the corresponding bands and extracted the DNA.

![](/wiki/images/2/26/T--Pittsburgh--data_week11_pcr.jpg)

Insertion bands

Top: pVS(28,29,30,31)

Bottom: pVS(33,34,43)

Fri (7/26/2019) Expression of pVS(29,30,34,43)

Victor, Ripal, Jemy, Mel

We expressed 250mL cultures of pVS(29,30,34,43) and pelleted to prepare for further purification.

Sat (7/27/2019) Purification of pVS(30,34,43)

Victor, Ripal, Jemy, Mel

We attempted Ni-NTA purification of three constructs and finally figured out the protocol.

All three constructs expressed and showed up in the elution with minimal contaminants.

![](/wiki/images/f/f3/T--Pittsburgh--data_week11_purification_30_34.png)

Ni-NTA purification of pVS30 and pVS34

![](/wiki/images/7/7a/T--Pittsburgh--data_week11_purification_43.png)

Ni-NTA purification of pVS43

## Week 12

Mon (7/29/2019) Purification of pVS29

Victor, Ripal, Jemy, Mel

![](/wiki/images/1/18/T--Pittsburgh--data_week12_purification_29.png)

Ni-NTA purification of pVS29

Tue (7/30/2019) Gibson assemblies + transformation of DH5α

Victor

We received gene fragments for set 3 from Twist and cloned them into pTEV5.
Also, we took the extracted inserts of set 1 and cloned them into pTEV6 to express an N-terminal MBP fusion protein.

Wed (7/31/2019) Colony PCR verification

Victor

Inserts were verified via colony PCR.

Thu (8/1/2019) Purification of pVS22 and pVS23

Ripal, Jemy

![](/wiki/images/f/f2/T--Pittsburgh--data_week12_purification_22_23.png)

Ni-NTA purification of pVS22(left) and pVS23(right)

pVS22 seems to be relatively insoluble.

## Week 13

Mon-Fri (8/5/2019)-(8/9/2019) Expression and purification of set 2 + pVS29

Harrison, Ripal

We were able to purify all the cosntructs with varying levels of purity.

U=uninduced, I=induced, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

![](/wiki/images/4/4b/T--Pittsburgh--data_week13_annotated_purification_20_26.png)

Ni-NTA purification of pVS20 and pVS26 (SDS-PAGE)

![](/wiki/images/5/54/T--Pittsburgh--data_week13_annotated_purification_22_23.png)

Ni-NTA purification of pVS22 and pVS23 (SDS-PAGE)

![](/wiki/images/0/01/T--Pittsburgh--data_week13_annotated_purification_24_25.png)

Ni-NTA purification of pVS24 and pVS25 (SDS-PAGE)

![](/wiki/images/b/b6/T--Pittsburgh--data_week13_annotated_purification_29.png)

Ni-NTA purification of pVS29 (SDS-PAGE)

Tue (8/8/2019) Retry colony PCR of pTEV6 constructs + set 3

Harrison, Ripal

After observing no colonies growing on AMP+ LB plates, we decided to retry colony PCR to verify the insertion.

Unfortunately, we didn't see a single positive hit from these colonies, indicating that cloning did not work.

![](/wiki/images/4/4c/T--Pittsburgh--data_week13_annotated_colony_pcr_36_37.png)

Colony PCR of pVS36 and pVS37 (+pVS21)

![](/wiki/images/9/95/T--Pittsburgh--data_week13_annotated_colony_pcr_38_39.png)

Colony PCR of pVS38 and pVS39 (+pVS21)

![](/wiki/images/6/6b/T--Pittsburgh--data_week13_annotated_colony_pcr_40_41.png)

Colony PCR of pVS40 and pVS41

![](/wiki/images/4/48/T--Pittsburgh--data_week13_annotated_colony_pcr_48_50.png)

Colony PCR of pVS48 and pVS50

Thu (8/10/2019) Lysate tests

Harrison, Ripal

We attempted to mix whole-cell lysates containing each of our intein halves to try to observe intein splicing.

However, background cellular proteins made it very difficult to identify any bands that were formed or depleted.

In the future, we could try using a His-tag stain to identify our synthetic protein constructs (and reactants).

Fri (8/11/2019) Sequencing of pVS28

Harrison

Miniprepped two other colonies of pVS28 and sent in for sequencing.

## Week 14, 15, 16

Everyone is on break

## Week 17

Wed (9/4/2019) Test purification with BugBuster

Ripal, Jemy

Attempted purification of pVS33 with BugBuster.

Protein did not show up in the elution.

![](/wiki/images/b/bc/T--Pittsburgh--data_week17_pVS33.png)

Purification of pVS33 (SDS-PAGE)

## Week 18

Mon (9/9/2019) PCR cloning of set 1 + Gibson assemblies

Victor

Attempted Gibson cloning of our set 1 constructs in pTEV6 and verified insertions via colony PCR.

Mon (9/9/2019) Transformation of CSL constructs

Ripal

Transformed several constructs from CSL into BL21 for expression.

Wed (9/11/2019) Transformed DH5α

Victor

Gibson assemblies were transformed into DH5α comp cells.

Fri (9/13/2019) Made BL21 comp cells

Victor

Prepared more BL21 comp cells for future transformations.

Sat (9/14/2019) Transformation + colony PCR of sspDnaB iGEM blocks

Ripal, Jemy

Two constructs encoding sspDnaB split-inteins were transformed into E. coli BL21 for expression and colony PCR was used to verify inserts.

![](/wiki/images/a/a4/T--Pittsburgh--data_week18_colony_pcr.png)

Colony PCR

Sat-Sun (9/14/2019)-(9/15/2019) Test purification with BugBuster

Ripal, Jemy

Attempted purification of pVS29 and pVS30 with BugBuster and sonic dismembrator as a control.

Gel overstained, it is difficult to tell which method worked better.

## Week 19

Mon (9/16/2019) Miniprepped all constructs + re-transformation of DH5α

Victor

Plates containing our existing constructs were transported to our secondary workspace in Dr. Childer's lab in order to make use of their equipment.
These constructs were miniprepped and retransformed into DH5α comp cells.

Wed (9/18/2019) DH5α & BL21 transformations

Victor

DH5α constructs from 9/16 were miniprepped and transformed into BL21 for expression.

Additonally a second set of transported constructs was miniprepped and transformed into DH5α.

Thu (9/19/2019) Remaining BL21 transformations

Victor

The second set of transported constructs was minipreppe from DH5α colonies and transformed into BL21 comp cells.

Thu (9/19/2019) Cloning of split-linker set and transformation into DH5α

Victor, Harrison

We received gBlocks encoding our split-linker constructs from IDT and cloned them into a pTEV6 backbone.
Gibson assemblies were transformed into E. coli DH5α cells and plated on LB media.
We also plated several previous assemblies of our pTEV6 variants of set 1.

Fri (9/20/2019) Freezer stock

Victor

Made freezer stock of all the transported BL21 colonies for future expression.

Fri (9/20/2019) Colony PCR

Harrison

Performed colony PCR on the newly transformed constructs in order to screen for insertions.

Used pTEV6 primers provided by a graduate student in Dr. Childer's lab.

Bold/underlined colonies indicate positive hits.

![](/wiki/images/f/f4/T--Pittsburgh--data_week19_pcr1.png)

Colony PCR 1

![](/wiki/images/c/c4/T--Pittsburgh--data_week19_pcr2.png)

Colony PCR 2

Sat (9/21/2019) Purification of pVS34 and pVS43

Ripal, Jemy

![](/wiki/images/8/8d/T--Pittsburgh--data_week19_purification_34_43.png)

Purification of pVS34 (left) and pVS43 (right) (SDS-PAGE)

## Week 20

Mon (9/23/2019) Miniprepped positive colonies

Harrison

Overnight colonies of the positive hits were miniprepped to prepare for sequencing.

Wed (9/25/2019) Ordered pTEV6 screening/sequencing primers

Harrison

Designed and ordered four new pTEV6 screening and sequencing primers to get better colony PCR results and prepare for Sanger sequencing.

Fri (9/27/2019) Colony PCR test with new primers

Harrison

Received primers from IDT and tested each combination of F/R primers on four constructs of pHG44.

All colonies showed strong positive hits.

![](/wiki/images/a/a9/T--Pittsburgh--data_week20_primer_test.png)

Colony PCR

Sat (9/28/2019) Repeat colony PCR

Harrison

Redid colony PCR of the previous constructs using the new pTEV6 primers.

![](/wiki/images/b/b5/T--Pittsburgh--data_week20_pcr1.png)

Colony PCR 1

![](/wiki/images/7/79/T--Pittsburgh--data_week20_pcr2.png)

Colony PCR 2

Sun (9/29/2019) Miniprepped and transformed BL21

Harrison

All the colonies with positive hits were miniprepped from overnight cultures and transformed into E. coli BL21 for expression.

![](/wiki/images/4/41/T--Pittsburgh--data_week20_bl21.jpg)

BL21 plates

## Week 21

Mon (9/30/2019) Expression of pVS22

Victor

Large scale expression of pVS22 for lysate mixture tests.

Tue (10/1/2019) Freezer stock of new constructs

Harrison

Created freezer stock of newly transformed BL21 constructs from overnight cultures.

Tue (10/1/2019) Small scale expression tests

Harrison

Grew 4mL cultures of the new constructs to OD600=0.5-0.6 and induced with 1mM IPTG for 4 hours.

All the constructs expressed protein at the expected size.

L=ladder, U=uninduced, I=induced

![](/wiki/images/4/47/T--Pittsburgh--data_week21_annotated_expr_test_1.png)

Expression test (SDS-PAGE)

![](/wiki/images/9/98/T--Pittsburgh--data_week21_annotated_expr_test_2.png)

Expression test (SDS-PAGE)

Wed (10/2/2019) Expression of pVS23

Victor

Large scale expression of pVS23 for lysate mixture tests.

Fri (10/4/2019) His stain test + lysate assay of pVS22/pVS23

Victor

We received a his-stain that can be applied to SDS-PAGE gels to visualize His-tagged proteins.

We tested the stain on a lysate mixture of pVS22 and pVS23.

Unfortunately we were unable to get access to the gel imager over the weekend and the gel destained considerably leaving faint bands.

![](/wiki/images/b/b2/T--Pittsburgh--data_week21_lys_22_23.png)

Lysate mixture of pVS22 and pVS23 (Coomassie brilliant blue) (SDS-PAGE)

![](/wiki/images/e/e2/T--Pittsburgh--data_week21_his_22_23.png)

Lysate mixture of pVS22 and pVS23 (InVision His-tag stain) (SDS-PAGE)

Sun (10/6/2019) Colony PCR of sspDnaB

Mel, Jemy

Ran colony PCR screening on several sspDnaB constructs from the 2019 iGEM distribution kit.

## Week 22

Mon (10/7/2019) Expression test of pHG68

Harrison

Tried expressing pHG68 at room temperature and 37 C to see if it would automatically splice like a cis intein.

While the construct expressed at the right size, we did not observe band formation corresponding to the spliced product.

On this gel we also tested diafiltration of pVS30 (E=elution, Df=after buffer exchange) to ensure our protein was retained during buffer exchange.

![](/wiki/images/0/07/T--Pittsburgh--data_week22_pHG68.png)

pHG68 expression test (SDS-PAGE)

Mon (10/7/2019) Large scale expression of pHG(44,52,60,64)

Harrison

Grew 200mL cultures of pHG(44,52,60,64) to OD600=0.8-0.9 and induced with 1mM IPTG for 4 hours.
Cultures were pelleted down and stored in -80 C for purification.

Mon (10/7/2019) Test diafiltration of pVS29

Ripal

Tested the new diafiltration columns on pVS29 to concentrate and exchange buffers.

Wed (10/9/2019) Expression of pVS47

Victor

Large scale expression of pVS47 for lysate mixture tests.

Wed (10/9/2019) Gibson assemblies

Victor

Redid Gibson assemblies of constructs that failed colony PCR screening from the third nested intein set and the split-linker set.

Wed (10/9/2019) Purification of pHG(44,52,60,64)

Harrison

Purified 200mL cultures of pHG(44,52,60,64) from frozen pellets.

All contructs except for pHG60 purified well.

pHG44 also has a relatively thick band in the elution below the expected size which we believe is a degredation product containing maltose binding protein.

U=uninduced, I=induced, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

![](/wiki/images/f/f5/T--Pittsburgh--data_week22_purification_44_52.png)

Purification gel (SDS-PAGE)

![](/wiki/images/1/19/T--Pittsburgh--data_week22_purification_60_64.png)

Purification gel (SDS-PAGE)

Wed-Fri (10/9/2019)-(10/11/2019) Expression, purification and diafiltration of pVS48

Ripal, Jemy, Mel

Large scale expression of pVS48 followed by purification and diafiltration.

Thu (10/10/2019) Diafiltration of pHG(44,52,60,64)

Harrison

Elutions of the four constructs were concentrated in centrifugal diafiltration columns and resuspended in a splicing buffer with high glycerol content intened to mimick the intracellular environment.

Thu (10/10/2019) In-vitro assay screen

Harrison

Several of the constructs were mixed in pairwise in-vitro assays to see if splicing would occur.

We were able to observe splicing between pHG44 and pHG64 however the concentration of pHG52 and pHG60 was too low to show up clearly on a gel.

![](/wiki/images/3/39/T--Pittsburgh--data_week22_iv_44_64.png)

Splicing assay (SDS-PAGE)

Important reactants and products are annotated

![](/wiki/images/d/d4/T--Pittsburgh--data_week22_iv_52_64.png)

Splicing assay (SDS-PAGE)

![](/wiki/images/7/7e/T--Pittsburgh--data_week22_iv_60_64.png)

Splicing assay (SDS-PAGE)

Fri (9/11/2019) Diafiltraion of pVS31

Mel, Ripal

Sat (10/12/2019) Large scale expression of pHG(44,59,60,68)

Harrison

Grew 200mL cultures of pHG(44,59,60,68) to OD600=0.7-0.8 and induced with 1mM IPTG at room temperature for 4 hours.

Cells were pelleted and stored in -80 C for purification.

Sat (10/12/2019) In-vitro kinetic measurements

Harrison

After observing splicing between pHG44 and pHG64, we ran another gel and took aliquots at certain time intervals to develop an understanding of the reaction rate.

We also ran both starting constructs at t=4 hours as negative controls to ensure we were not seeing degredation products.

![](/wiki/images/1/1a/T--Pittsburgh--data_week22_kin_44_64.png)

Splicing kinetic assay (SDS-PAGE)

Important reactants and products are annotated

Sun (10/13/2019) Transformation and colony PCR of BBa\_K590016

Victor

Transformed a construct from the 2019 iGEM distribution kit for characterization.

## Week 23

Mon (10/14/2019) Expression of pVS47 and pVS48

Victor

Large scale expression of pVS47 and pVS48 for lysate mixture tests.

Mon (10/14/2019) Purification of pHG(44,59,60,68)

Harrison

Purified 200mL cultures of pHG(44,59,60,68). All constructs purified well.

U=uninduced, I=induced, P=pellet, S=supernatant, Ft=flow-through, W=wash, E=elution

![](/wiki/images/6/64/T--Pittsburgh--data_week23_purification_44_59.png)

Purification gel (SDS-PAGE)

![](/wiki/images/0/02/T--Pittsburgh--data_week23_purification_60_68.png)

Purification gel (SDS-PAGE)

Tue (10/15/2019) Expression of pVS45 and pVS29

Victor

Large scale expression of pVS45 and pVS29 for lysate mixture tests.

Tue (10/15/2019) In-vitro screening of new constructs

Harrison

Performed overnight in-vitro assays of several combinations of the recently purified constructs.

We observed splicing between pHG44 and pHG64 (as seen before) and also a hint of splicing between pHG60 and pHG64 (predicted).

However, we did not observe splicing between pHG59 and pHG44 as expected. After carefully reviewing our construct sequences, we realized that our design was missing a single amino acid residue in gp41-1 N.

![](/wiki/images/e/e3/T--Pittsburgh--data_week23_multi.png)

In-vitro assay screening (SDS-PAGE)

The four constructs on the left are at t=0

For each reaction mixture, the left lane is t=0 and the right lane is t=20 hours at 37 C


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# Team:Pittsburgh/Experiments

[Pitteins](/Team%3APittsburgh)

[Team](/Team%3APittsburgh/Team)

[Project](/Team%3APittsburgh/Description)

[Modeling](/Team%3APittsburgh/Model)

[Human Practices](/Team%3APittsburgh/Human_Practices)

[Safety](/Team%3APittsburgh/Safety)

[Team](/Team%3APittsburgh/Team)

[Attributions](/Team%3APittsburgh/Attributions)

[Collaborations](/Team%3APittsburgh/Collaborations)

[Description](/Team%3APittsburgh/Description)

[Design](/Team%3APittsburgh/Design)

[Parts](/Team%3APittsburgh/Parts)

[Experiments](/Team%3APittsburgh/Experiments)

[Notebook](/Team%3APittsburgh/Notebook)

[Contribution](/Team%3APittsburgh/Contribution)

[Results](/Team%3APittsburgh/Results)

[Human Practices](/Team%3APittsburgh/Human_Practices)

[Public Engagement](/Team%3APittsburgh/Public_Engagement)

## Pittsburgh iGEM 2019 Experimental Protocols

TABLE OF CONTENTS

[Buffers & Reagents](#h.do6i9qbjts44)

[Antibiotic Concentrations](#h.qlsftg9jaeez)

[Protein Buffers](#h.e9m8l7uds3v0)

[Storage Buffer (Protein Purification)](#h.u0q6q4jbehn7)

[Resuspension Buffer (Protein Purification)](#h.gux7uib6hrmu)

[Lysis Buffer (Protein Purification)](#h.up4bw92p9ezf)

[Wash Buffer (Protein Purification)](#h.huq97jonkefz)

[Elution Buffer (Protein Purification)](#h.55ui1ap6e4d9)

[Dialysis Buffer (Protein Purification)](#h.jbhhx2mj6dn6)

[Splicing Buffer (Intein Assays)](#h.ujpcrj5avst3)

[Mootz Splicing Buffer (Intein Assays)](#h.enw7zm8hd82d)

[1x SDS Running Buffer](#h.3hcddfxqf3t4)

[5X ISO Buffer](#h.cx3lnbors5cy)

[Coomassie Stain for Protein Gels](#h.v1921ueo8slr)

[5X KCM](#h.b74jjdmq4xmk)

[Denaturing SDS Sample Buffer (3x)](#h.hbr00fgz25t)

[Transformation Storage Buffer (TSB)](#h.jh4s3stjebte)

[Preparation of Competent Cells](#h.o4xmwl76wl0n)

[Day 1:](#h.ndyzqoue66xw)

[Day 2:](#h.7zeh29srgc7i)

[Cloning](#h.nftcq7m2rsq4)

[Phusion Polymerase](#h.z83nca6dkx0d)

[Q5 2x Master Mix](#h.lejjgfdm7fcb)

[DreamTaq 2x Master Mix](#h.lphq5nlciqu7)

[DNA Gel Electrophoresis](#h.luxoqfqx62kh)

[Gibson Cloning](#h.5cmzsh5cks4s)

[Transformation](#h.rhu66sl5213j)

[Transformation](#h.3xocfox5d522)

[Miniprep](#h.xn7xiaj3gnpo)

[Plasmid Screening (Colony PCR)](#h.b76bd39bvuj7)

[Microscope preparation (basic compound microscope)](#h.g1eostuivki)

[Protein Expression](#h.80c4d6b781zm)

[Expression Optimization Protocol](#h.2p6ap0fwbllm)

[Protein Purification](#h.w43zne65x3a3)

[Purification Optimization Protocol](#h.i5yp1a7o9jgp)

[Day I](#h.4kwix5qmytky)

[Gel Staining](#h.6y6hkrlzvn9f)

[Coomassie Staining](#h.1mpewzq1eiay)

[Coomassie Destaining](#h.cq1btrl758vg)

[Diafiltration -Buffer Exchange](#h.fjyvw94rf1bu)

[Intein Assays](#h.2s9ovveuoyzn)

[Purified and Dialyzed Protein Assays](#h.l0tiqsx4f6fg)

[Lysate Splicing Assays](#h.hrd74kyawdmn)

---

#

# Buffers & Reagents

## Antibiotic Concentrations

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | | | Volume of antibiotic to add in microliters per | |
| Antibiotic | Stock Solution Name | Stock Concentration (mg/mL) | LB plates (20mL/plate) | LB Broth  (1 mL) |
| Kanamycin | Kan 30 | 30 | 33.33 | 1 |
| Chloramphenicol | Chlor 30 | 30 | 20 | 0.67 |
| Streptomycin | Strep 50 | 50 | 12 | 0.60 |
| Ampicillin | Amp 100 | 100 | 20 | 0.50 |

## Protein Buffers

### Storage Buffer (Protein Purification)

Total volume 4 L

* 150 mM NaCl
* 20 mM TrisHCl pH 7.4
* 20% Glycerol
* 1 mM β-mercaptoethanol (Add right before using)

### Resuspension Buffer (Protein Purification)

Total volume 1 L

* 50 mM HEPES, pH 8.0
* 150 mM NaCl

### Lysis Buffer (Protein Purification)

100 mL, store at 4 ̊C

* 150 mM NaCl
* 20 mM Tris Base, pH 7.4
* 20 mM imidazole, pH 7.0
* 1 mM β-mercaptoethanol

* Add right before using

* 1 EDTA-free protease inhibitor tablets

* Add right before using

* 100 U DNase

* Add right before using

* 0.1% Triton-X 100

* Add right before using

### Wash Buffer (Protein Purification)

Total volume 250 mL

* 150 mM NaCl
* 20 mM Tris Base, pH 7.4
* 40 mM imidazole, pH 7.0
* 1 mM β-mercaptoethanol

* Add right before using

### Elution Buffer (Protein Purification)

Total volume 250 mL

* 150 mM NaCl
* 20 mM Tris Base, pH 7.4
* 200 mM imidazole, pH 7.0
* 1 mM β-mercaptoethanol

* Add right before using

### Dialysis Buffer (Protein Purification)

Total volume 1 L

* 150 mM NaCl
* 20 mM Tris Base, pH 7.4
* 1 mM β-mercaptoethanol

* add right before using

### Splicing Buffer (Intein Assays)

Total volume 250 mL

* 150 mM 5 M NaCl
* 20 mM 2 M Tris Base, pH 7.4
* 1 mM β-mercaptoethanol

* Add right before using

* Protease inhibitor tablet

* Add right before using

* 10% (v/v) Glycerol

### Mootz Splicing Buffer (Intein Assays)

Stored in aliquots at -80°C

* 50 mM Tris-HCl at pH 7.0
* 300 mM NaCl
* 1 mM EDTA
* 10% (v/v) glycerol
* 2 mM DTT

### 1x SDS Running Buffer

Total volume 1L

* 14.4 g Glycine
* 3.0 g Tris Base
* 1 g SDS

## 5X ISO Buffer

Store at -20°C in 320 µL aliquots

* 3 mL of 1M Tris Base at pH 7.5
* 150 µL of 2 M MgCl2
* 240 µL of 100 mM dNTP mix
* 300 µL of 1 M DTT
* 1.5 g PEG-8000
* 600 µL of 50 mM NAD+
* Fill to 6 mL with deionized water

##

## Coomassie Stain for Protein Gels

1 L, store in the dark at room temperature

* 60-80 mg Coomassie G-250
* 1 L MilliQ water

* Stir for several hours to dissolve

## 5X KCM

30 mL total. Sterile filter, store at 4°C

* 0.5 M KCl
* 0.15 M CaCl2
* 0.25 M MgCl2

##

## Denaturing SDS Sample Buffer (3x)

10 mL total. Store at -20°C

* 1.5 mg Bromophenol Blue
* 0.60 g SDS
* 3.0 mL Glycerol
* 3.9 mL of 0.5 M Tris Base at pH 6.8
* 1.5 mL of β-mercaptoethanol

## Transformation Storage Buffer (TSB)

50 mL total. Sterile filter and store at 4°C

* 20.5 mL LB broth at pH 6.5
* 10% PEG-3350
* 5% DMSO
* 10 mM MgCl2
* 10 mM MgSO2

# Preparation of Competent Cells

## Day 1:

1. Grow a 5 mL LB culture from a freezer stock of the strain of cells at 37°C overnight

## Day 2:

1. Innoculate 100 mL LB media with 1mL of the culture from the day before

2. Shake at 37°C to OD600=0.4-0.6
3. Place culture on ice for 5 min
4. Pellet cells at 4000 xg for 10 minutes
5. Resuspend cells in 10 mL of chilled TSB
6. Incubate on ice for 5 minutes
7. Subpackage ino tubes (100 µL aliquots) and freeze in liquid nitrogen
8. Store at -80°C

---

#

# Cloning

## Phusion Polymerase

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PCR Component | Number of PCR Reactions\* | | | | | | |
| 1 | 5 | 9 | 11 | 15 | 16 | 21 |
| 5X GC Buffer | 10 | 50 | 90 | 110 | 150 | 160 | 210 |
| dNTPs (10mM) | 1 | 5 | 9 | 11 | 15 | 16 | 21 |
| PCR Grade Water | 20 | 100 | 180 | 220 | 300 | 320 | 420 |
| DMSO | 1.5 | 7.5 | 13.5 | 16.5 | 22.5 | 24 | 31.5 |
| Betaine (5 M) | 13 | 65 | 117 | 143 | 195 | 208 | 273 |
| Sense Primer (10 µM) | 1.5 | 7.5 | 13.5 | 16.5 | 22.5 | 24 | 31.5 |
| Antisense Primer (10 µM) | 1.5 | 7.5 | 13.5 | 16.5 | 22.5 | 24 | 31.5 |
| Template DNA | 1 | 5 | 9 | 11 | 15 | 16 | 21 |
| Phusion DNA Polymerase (1 U/µL) | 0.5 | 2.5 | 4.5 | 5.5 | 7.5 | 8 | 10.5 |
| Total Volume | 50 | 250 | 450 | 550 | 750 | 800 | 1050 |

\*amounts for each are in uL

## Q5 2x Master Mix

Q5 Master Mix recipe available on New England Biolabs Website

## DreamTaq 2x Master Mix

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PCR Component | Number of PCR Reactions\* | | | | | | |
| 1 | 5 | 9 | 16 | 21 | 26 | 31 |
| 2X Master Mix (DreamTaq) | 5 | 25 | 45 | 80 | 105 | 130 | 155 |
| Forward Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
| Reverse Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
| Betaine (5 M) | 2.6 | 13 | 23.4 | 41.6 | 54.6 | 67.6 | 80.6 |
| DMSO | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
| Water | 0.9 | 4.5 | 8.1 | 14.4 | 18.9 | 23.4 | 27.9 |
| Total Volume | 10 | 50 | 90 | 160 | 210 | 260 | 310 |

\*amounts for each are in uL

|  |  |  |
| --- | --- | --- |
| Step | Temp | Time |
| Initial Denaturation | 98°C | 30 seconds |
| 25 cycles | 98°C  45-72°C  72°C | 5-10 seconds  10- 30 seconds  60- 120 seconds per kb |
|
|
| Final Extension | 72°C | 5-10 minutes |
| Hold | 4°C |  |

## DNA Gel Electrophoresis

Preparing Gel

* Weigh 1 g of ultrapure agarose. Add to 100 mL of 1x TAE buffer solution
* Microwave and swirl periodically until fully dissolved
* When almost cooled, add 2 µL of ethidium bromide
* Place combs, desired side down, and pour solution

Running Gel

* Pour 1x TAE buffer till entire gel is covered
* Load samples

* In the first well, load 10 µL of desired DNA ladder
* Amount of PCR product depends on comb used to create well

* 50 µL of pcr product
* 10 µL of 6x loading dye

* Mix by pipetting

* Plug in the electrodes in a Negative to Positive direction

* Check for bubbling at the negative end (top)

* Run for 25 minutes at 125V
* Turn off electrodes before removing cover
* Look at gel under UV transilluminator

* Compare size of band against ladder

* Use a razor blade to cut and transfer band of DNA to a clean, pre-labeled eppendorf tube
* Repeat for each well.
* Freeze at -20°C or continue onto gel purification protocol
* Dispose of gel into specified ethidium bromide waste

##

## Gibson Cloning

* Prepare Gibson Assembly Master Mix as follows:

* Store at -20°C in 15 µL aliquots

|  |  |
| --- | --- |
| Component | Volume (uL) |
| 5X ISO Buffer | 320 |
| 10 U/µL T5 exonuclease | 0.64 |
| 10 U/µL Phusion polymerase | 20 |
| 40 U/µL Taq ligase | 160 |
| Water | 699 |
| Total | 1200 |

* Thaw a 15 µL aliquot of the Gibson assembly master mix and keep on ice until use
* Measure the DNA concentration (ng/µL) of each assembly piece
* Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to the thawed 15 µL master mix in a 20 µL total volume assembly reaction mixture as follows:

|  |  |  |
| --- | --- | --- |
|  |  | Linearized vector backbone (100 ng) |
| + |  | Each additional assembly piece (to equimolar with backbone) |
| + | 15 µL | Gibson assembly master mix |
| + |  | dH2O to |
|  | 20 µL |  |

* Incubate the assembly reaction at 50°C for 60 minutes

* Store at -20°C
* Use for transformations into DH5α competent cells

  ---

# Transformation

## Transformation

1. Combine

1. 20 µL 5x KCM
2. + x µL DNA (~10 ng or 5uL of Gibson reaction product)
3. + ddH2O to 100 µL

2. Add 100 µL competent cells, mix gently and incubate on ice for 20 minutes

1. DH5α for Gibson products and plasmid storage
2. Bl21 for Protein Expression
3. Rosetta for rare codon protein expression

3. Heat shock at 42°C for 90 sec
4. Place on ice for 1 minute
5. Add 400-600 µL LB media to cells
6. Shake for 45 min to 1 hour at 37°C
7. Plate 50-250 µL cells

##

## Miniprep

* Transfer 1.5 mL of culture eppendorf tubes
* Centrifuge for 1 min to pellet cells

* Discard supernatant

* Repeat 2 times
* Using the ThermoFisher Genejet kit

* Add 250uL of resuspension buffer to the pellet and resuspend
* Pipet up and down to fully resuspend cells

* Should become cloudy

* Add 350uL of Lysis solution
* Invert 4-6 times

* Should turn clear and viscous

* Add 350uL of neutralization solution
* Invert 4-6 times

* White precipitate should form

* Centrifuge for 5 minutes at 14.7 xg to pellet cell walls and chromosomal DNA
* Transfer supernatant to centrifugal filter tubes
* Centrifuge for 1 minute
* Discard flow through

* Continue until all of the sample has been filtered

* Elute with sterile water into clean, labeled eppendorf tube.

* 50mL

##

## Plasmid Screening (Colony PCR)

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| PCR Component | 1 | 5 | 9 | 16 | 21 | 26 | 31 |
| 2X Master Mix (DreamTaq) | 5 | 25 | 45 | 80 | 105 | 130 | 155 |
| Forward Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
| Reverse Primer (10 µM) | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
| Betaine (5 M) | 2.6 | 13 | 23.4 | 41.6 | 54.6 | 67.6 | 80.6 |
| DMSO | 0.5 | 2.5 | 4.5 | 8 | 10.5 | 13 | 15.5 |
| Water | 0.9 | 4.5 | 8.1 | 14.4 | 18.9 | 23.4 | 27.9 |
| Total Volume | 10 | 50 | 90 | 160 | 210 | 260 | 310 |

|  |  |  |
| --- | --- | --- |
| Step | Temp | Time |
| Initial Denaturation | 98°C | 30 seconds |
| 35 cycles | 98°C  45-72°C  72°C | 5-10 seconds  10- 30 seconds  60- 120 seconds per kb |
|
|
| Final Extension | 72°C | 5-10 minutes |
| Hold | 4°C |  |

## Microscope preparation (basic compound microscope)

1. Add 10uL of sterile water to a pcr tube
2. Use a pipet-tip to transfer some cells from plate to the water by lightly scraping cells off the plate and swirling it in the water

1. Plate 2 µL of cells onto a microscope slide, place a slide cover over sample

   ---

# Protein Expression

## Expression Optimization Protocol

Day 1:

1. Inoculate specified volume\* of LB media + antibiotic overnight at 37°C in a 150 mL flask with BL21 cells with desired plasmid

\* 10 mL of overnight culture per 1 L of expression culture

Day 2:

2. Inoculate large culture of LB media + antibiotic with overnight culture

1. Ensure culture flask is at least 3x the volume of the culture to ensure adequate aeration

3. Grow to OD600 of 0.4 to 0.6
4. Take a 1 mL sample of the culture, centrifuge at 21,000 xg for 1 minute, and resuspend in 50 µL of denaturing sample buffer. Set aside for expression verification gel.
5. Induce expression with 1mM [IPTG]final

1. [IPTG]final = 1 mL of  1M IPTG per liter of large culture

6. Shake at 37°C for 4 hours
7. Take a 1 mL sample of the culture, centrifuge at 21,000 xg for 1 minute, and resuspend in 50 µL of denaturing sample buffer. This will be run with the 1 mL sample from earlier.
8. Heat both 1mL samples at 75-100 °C for 5-10 minutes.
9. Vortex and centrifuge again at 21,000 xg for 5 minute before loading on a gel to test expression
10. Spin large cultures down at 4000 xg for 15 minutes

1. Pour off liquid into sink
2. Repeat for all of the same culture into the same bottle

11. Resuspend in resuspension buffer.
12. Pellet resuspended cells into 50 mL tubes (conicals) at 4000 xg for 10 minutes
13. Pour off liquid and freeze at -80°C until ready for purification

---

#

# Protein Purification

## Purification Optimization Protocol

### Day I

1. Resuspend cells in the 20-100 mL Lysis Buffer and scrape, vortex, or pipette up and down.
2. Lyse the cells using a Sonic-Dismembrenator or Emulsiflex and collect the resulting solution.
3. Clarify the lysate by centrifugation at 20,000 xg at 4 °C for 90 minutes.

1. Take a small sample of the pellet and store in denaturing SDS buffer
2. Take a small sample of the supernatant and store in denaturing SDS buffer

4. Incubate supernatant with Ni-NTA resin for 2 hours
5. Transfer supernatant + Ni-NTA resin into a size-exclusion column and allow to drain

1. Take a small sample of the flowthrough and store in denaturing SDS buffer

6. Wash the column with Wash Buffer 3 times.

1. Take a small sample of the wash sample and store in denaturing SDS buffer

7. Elute the protein using Elution Buffer 1 times.

1. Take a small sample of the wash sample and store in denaturing SDS buffer

8. Analyze protein purity by SDS-PAGE.

1. Run all of the samples taken prior to check for presence of protein.

#

---

#

# Gel Staining

## Coomassie Staining

1. Microwave the gel in deionized water for 30 seconds. Repeat three times to remove any residual SDS.
2. Soak the gel in enough Coomassie Safe Stain to cover the surface, microwave for 30 seconds, and let sit for 10 minutes, shaking occasionally.

## Coomassie Destaining

3. Pouring out theCoomassie stain
4. Rinse the gel with deionized water
5. Cover the gel in deionized water and microwave the gel for 30 seconds
6. Leave on orbital shaker until gel is destained

1. Add a kimtech wipe to absorb residual coomassie stain.

---

# Diafiltration -Buffer Exchange

1. Load elution into diafiltration column with the column facing downwards
2. Centrifuge at 4000 xg for 45 min at 4°C to concentrate sample
3. Add 2 mL splicing buffer to the column.
4. Centrifuge at 4000 xg for 45 min at 4°C to concentrate and exchange buffers
5. Remove filter collection tube and invert column
6. Centrifuge at 1000 xg for 2 min at 4°C to elute sample
7. Store at -20°C

   ---

# Intein Assays

## Purified and Dialyzed Protein Assays

1. Thaw the splicing buffer dialyzed proteins on ice
2. Combine equimolar concentrations of each protein

1. Shake at 37°C

3. Take 2 uL aliquots at designated times and store samples in denaturing SDS buffer
4. Run gel

1. Run with individual proteins as controls

5. Stain gel with Coomassie Stain

## Lysate Splicing Assays

1. Lyse expressed pellets through physical means (sonication, emulsiflex) into splicing buffer
2. Set aside 2uL of lysate in denaturing SDS to be run on the gel
3. Optional centrifugation to clarify lysates (depends on protein solubility)
4. Combine each protein lysate

1. Shake at 37°C

5. Take 2 uL aliquots at designated times

6. Run gel

1. Run with individual lysates as controls

7. Stain gel with Coomassie Stain or HIS- Stain

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Pittsburgh/Results

[Pitteins](/Team%3APittsburgh)

[Team](/Team%3APittsburgh/Team)

[Project](/Team%3APittsburgh/Description)

[Modeling](/Team%3APittsburgh/Model)

[Human Practices](/Team%3APittsburgh/Human_Practices)

[Safety](/Team%3APittsburgh/Safety)

[Team](/Team%3APittsburgh/Team)

[Attributions](/Team%3APittsburgh/Attributions)

[Collaborations](/Team%3APittsburgh/Collaborations)

[Description](/Team%3APittsburgh/Description)

[Design](/Team%3APittsburgh/Design)

[Parts](/Team%3APittsburgh/Parts)

[Experiments](/Team%3APittsburgh/Experiments)

[Notebook](/Team%3APittsburgh/Notebook)

[Contribution](/Team%3APittsburgh/Contribution)

[Results](/Team%3APittsburgh/Results)

[Human Practices](/Team%3APittsburgh/Human_Practices)

[Public Engagement](/Team%3APittsburgh/Public_Engagement)

## Results

## Overview

**Nested intein system**

* Designed/synthesized/purified two sets of nested intein constructs
* Demonstrated proof-of-concept splicing of nested intein constructs *in-vitro*
* Characterized the (in)solubility of split-intein fusion proteins

**Split-linker system**

* Designed/synthesized/purified split-linker constructs
* Demonstrated the viability of intein-based linker construction *in-vitro*
* Demonstrated the orthogonality of split-inteins in small linker fragments

## Nested Intein Splicing Assays

![](/wiki/images/f/fc/T--Pittsburgh--figures_results_positive_nested1.png)

As a proof of concept test for our nested intein system, we did a splicing assay of BBa\_K3308083 (pVS00047) and BBa\_K3308084 (pVS00048). These plasmids contain the NrdJ-1 C-terminal split between the fourth and fifth amino acid by TvoVMA. Splicing of tvoVMA inteins together will reconstitute the NrdJ-1 C terminal intein. The sequences flanking the TvoVMA intein were the unmutated NrdJ-1 C terminal residues. As demonstrated by Figure 1, at 24 hours, intein splicing is observed by the presence of the splice product.

## Split-Linker Splicing Assays

![](/wiki/images/6/6a/T--Pittsburgh--figures_results_positive_split-linker.png)

Figure 2
Coomassie stained SDS-PAGE of the splice test of split linker constructs BBa\_K3308027 (pVS00044) and BBa\_K3308034 (pVS00064). Purified protein was mixed and incubated at 37 °C for 24 hours. Samples were taken at 5 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes and 1 hour and stored in denaturing SDS buffer. Also included on the gel are samples of the individual constructs at 0 seconds and 4 hours as well as the protein ladder.

As a proof of concept test for our Split-linker system, we did a splicing assay of BBa\_K3308027 (pVS00044)and BBa\_K3308034 (pVS00064). These plasmids contain the GP41-8 which forms the connection between the SceVMA intein and the linker. The sequences were the native flanking sequences for GP41-8. This allowed for rapid splicing to occur. A faint band is visible in the 5 second As demonstrated by Figure 1, at 24 hours, intein splicing is observed by the presence of the splice product.

## Expression Tests

![](/wiki/images/5/54/T--Pittsburgh--figures_results_annotated_expr_test_1.png)
![](/wiki/images/6/68/T--Pittsburgh--figures_results_annotated_expr_test_2.png)

Figure 3
These gels are Coomassie stained and show the positive expression of our constructs by inducing with 1 mM IPTG at OD600=0.6. Every other lane shows the uninduced sample as the control and the induced sample as the results.

After cloning our constructs into BL21, we tested our constructs to see if we were able to see the constructs at the correct size on the SDS-PAGE. As shown above, all of our constructs showed positive expression of all of our constructs.

![](/wiki/images/1/1b/T--Pittsburgh--figures_results_60_64.png)

Figure 4
This Coomassie stained gel shows the purification of BBa\_K3308034 (pVS00064). The lanes from left to right are uninduced, induced, pellet, supernatant, flow-through, wash, and elution.

As seen in this gel, the purification of BBa\_K3308034 (pVS00064) was relatively successful. There was a thick band that showed up in the elution lane of the gel. However, there seemed to be some nonspecific binding that showed up in the elution, resulting in some impurities in the gel. Our next step for this gel is the diafiltration in order to improve the purity and concentration of the protein.