Part:BBa_K3425001

pSB3C11: Improved pEven1 Loop Vector based on pSB3C01

iGEM Type IIS Vectors

pEven Loop Vectors (for Level 2)

When four Level 1 parts (TUs) are assembled into a Multi-Transcriptional Unit (MTU) into the following plasmid backbones (with BBa_J04455), the MTU will be flanked by BsaI restriction sites and these 4 bp Fusion Sites.

Part number	Name	Loop Name	Fusion Site 5'	MTU	Fusion Site 3'
BBa_K3425001	pSB3C11	pEven1	GGAG	MTU 1	TACT
BBa_K3425002	pSB3C12	pEven2	TACT	MTU 2	AATG
BBa_K3425003	pSB3C13	pEven3	AATG	MTU 3	GCTT
BBa_K3425004	pSB3C14	pEven4	GCTT	MTU 4	CGCT

The iGEM Type IIS assembly standard is based on MoClo and Loop

Note: This section was adapted from pSB3C01 in order to collect all the information in one page.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2713
Illegal PstI site found at 12
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2713
Illegal NheI site found at 1399
Illegal PstI site found at 12
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2713
23
INCOMPATIBLE WITH RFC[23]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2713
Illegal PstI site found at 12
25
INCOMPATIBLE WITH RFC[25]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2713
Illegal PstI site found at 12
Illegal AgeI site found at 990
Illegal AgeI site found at 1313
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 2719
Illegal BsaI.rc site found at 6

Mutagenesis of pSB3C0# pEven Loop vectors

The sequence of pEven Loop vectors for Level 2 assemblies (pSB1C0#) contains an additional, illegal BsaI site. BsaI is the enzyme which is used to cleave the assembly out of a pEven vector to be cloned back to a pOdd vector. Therefore, the presence of this illegal site would impact the construction of Level 3 assemblies. Site-directed Mutagenesis by PCR was conducted in order to remove the illegal sites. The new plasmids were named pSB3C1#. This page corresponds to pSB3C11 which was obtained from pSB3C01.

After PCR mutagenesis, screening was done by digestion of the plasmid DNA with BsaI (Figure 1). All backbones (pSB3C0# and pSB3C1#) contain a band that corresponds to the mRFP1 cassette (BBa_J04455) with a size of 1093bp. Then, pSB3C1# consist of one more band of 2725pb. Since pSB3C0# have one more BsaI site, they should present two more bands of 2434bp and 291bp. While the 291bp band is not visible, probably due to too low DNA amount, the shift in size before and after mutagenesis is clearly visible.

Mutagenised Type IIS Level 2 backbones — Figure 1. 1% Agarose Gel Electrophoresis - SYBR Safe analysis of BsaI digested plasmids. Linearised plasmid and non-digested plasmid were also loaded in order to interpret unexpected bands correctly.

This difference cannot be seen, however, for pSB3C01 and pSB3C11. In addition, a longer band can be seen for pSB3C01, but a comparison to well 10 shows that it corresponds to linearised plasmid. To investigate the lack of difference between pSB3C01 and pSB3C11, as well as to confirm the mutagenesis by another method, pSB3C01 and pSB3C1# were sequenced.

Sequencing results showed a successful PCR mutagenesis for all of the pSB3C1# backbones, with the expected point mutation. Furthermore, they also showed a point mutation in pSB3C01 (at a different point than the planned one) which eliminated the extra BsaI site.

Afterwards, some assemblies into pSB3C1# were conducted, which show their functionality. This confirmed that these backbones would work as the new pEven set of vectors.

For further information on this experiment along with the sequencing results, please visit Team Uppsala 2020's Wet lab page.

insert	RFP-device (J04455)
origin	p15A
resistance	C